scholarly journals Modulation of expression of endogenous collagenase and collagen genes by electroporation: possible involvement of Ca2+ and protein kinase C

1993 ◽  
Vol 290 (1) ◽  
pp. 135-138 ◽  
Author(s):  
C A Lambert ◽  
P Y Lefebvre ◽  
B V Nusgens ◽  
C M Lapière
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Hela Cells ◽  
Mrna Level ◽  
Dermal Fibroblasts ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Kinase C ◽  
Voltage Dependent ◽  
Collagen Genes

We have investigated the effect of electroporation on the expression of collagen alpha 1(I), collagenase, c-fos and c-jun genes in human dermal fibroblasts (HDF), human smooth muscle cells (HSMC) and HeLa cells. Collagenase and collagen mRNA levels were respectively increased and decreased in a voltage-dependent manner in HDF harvested 2 days after a sham electroporation. These effects were still observed 10 days after electroporation. Similar effects occurred in electroporated HSMC. Neither collagen nor collagenase mRNAs were detected in control or electroporated HeLa cells. c-fos and c-jun mRNA levels were also increased in electroporated HDF, HSMC and HeLa cells harvested 1 h after plating. This suggests that factor AP1 (fos/jun) could mediate the up-regulation of collagenase expression in electroporated HDF and HSMC. When electroporation of HDF was performed in the presence of H7, an inhibitor of protein kinase C, no increase in collagenase mRNA level was observed, suggesting that protein kinase C might be involved in the transduction of the effect. All the effects reported were also suppressed when cells were electroporated in a medium containing EGTA, suggesting that Ca2+ might mediate the transduction of this effect.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

scholarly journals Post-transcriptional regulation of apolipoprotein E expression in mouse macrophages by phorbol ester

1993 ◽  
Vol 292 (1) ◽  
pp. 105-111 ◽  
Author(s):  
L Dory
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Apolipoprotein E ◽  
Phorbol Ester ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Mouse Macrophages ◽  
Apoe Expression

Phorbol ester-mediated differentiation of THP-1 cells (a human monocytic cell line) into mature macrophages is associated with a transcriptional induction of apolipoprotein E (apoE) expression [Auwerx, Deeb, Brunzell, Peng and Chait (1988) Biochemistry 27, 2651-2655]. Endotoxin, on the other hand, which may also act through activation of protein kinase C, is a potent inhibitor of apoE expression in mouse macrophages [Werb and Chin (1983) J. Biol. Chem. 258, 10642-10648]. The present experiments examine the effect of phorbol ester, an activator of protein kinase C, on the apoE expression in mouse thioglycollate-elicited peritoneal macrophages. Phorbol ester inhibits apoE expression in a specific, time- and dose-dependent manner. A 75% inhibition in the rate of apoE secretion, but not that of total protein, was observed following a 4.5 h incubation with 160 nM phorbol ester, although nearly full inhibition was obtained with 40 nM. The changes in apoE secretion were paralleled by similar changes in apoE synthesis, indicating synthesis as the primary site of action. The decreased rates of apoE synthesis are shown not to be due to increased apoE degradation. The profound inhibition of apoE synthesis was not accompanied by significant changes in apoE mRNA levels at any concentration of phorbol ester (up to 16 microM), or length of treatment (up to 24 h), suggesting a post-transcriptional locus of regulation of apoE expression. Although the early changes in apoE synthesis correlate with increased microsomal protein kinase C activity, the suppression of apoE expression persists even during conditions of nearly complete (> 95%) loss of protein kinase C activity, suggesting that the direct or indirect effect of protein kinase C on apoE expression is mediated by a stable phosphorylated protein, or that the observed effects are mediated through a protein kinase C species that is not readily downregulated by phorbol esters. The presented studies clearly demonstrate the potential importance of the translational regulation of apoE expression through the protein kinase C signal transduction pathway.

scholarly journals Transcriptional Activation of the Ovine Follicle-Stimulating Hormone β-Subunit Gene by Gonadotropin-Releasing Hormone: Involvement of Two Activating Protein-1-Binding Sites and Protein Kinase C**This work was supported by the North Carolina State University Agricultural Research Service, NICHD Grant 34863, and the Mellon Foundation.

Endocrinology ◽  
1998 ◽  
Vol 139 (11) ◽  
pp. 4455-4465 ◽  
Author(s):  
Brian D. Strahl ◽  
Huey-Jing Huang ◽  
Joseph Sebastian ◽  
Basavdutta R. Ghosh ◽  
William L. Miller
Keyword(s):  
Transcriptional Regulation ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Hela Cells ◽  
Cell System ◽  
Proximal Promoter ◽  
Dependent Manner ◽  
Β Subunit ◽  
Subunit Gene ◽  
Kinase C

Abstract FSH is an α/β heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its β-subunit. Recent studies on transcriptional regulation of the ovine FSH β-subunit gene (oFSHβ) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at −120 and −83 bp) that are probably physiologically important for FSHβ expression. As GnRH is a major regulator of FSHβ expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSHβ transcription may be regulated by GnRH through AP-1. Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHβ. This system was used to show that expression of luciferase constructs containing either the −4741/+759 region of the oFSHβ gene (−4741oFSHβ-Luc) or the −846/+44 region of the human α gene (α-Luc; a positive control) was stimulated 3.1 ± 0.3- and 7.7 ± 1.9-fold, respectively, by 100 nm GnRH. Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH. Similar results with these constructs were obtained in COS-7 cells. Studies with progressive 5′-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSHβ. Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity. Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nm) and GnRHR, which was identified by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSHβ and α-subunit genes by GnRH in HeLa cells was investigated. Although 12-O-tetradecanoyl 13-acetate induction of α-Luc and −215oFSHβ-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57–65% of the GnRH-mediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction. These data define a molecular action of GnRH on oFSHβ gene transcription that involves two proximal AP-1 enhancer elements and PKC activation. Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells.

Substance P potentiates calcium channel modulation by somatostatin in chick sympathetic ganglia

1994 ◽  
Vol 72 (6) ◽  
pp. 2683-2690 ◽  
Author(s):  
A. Golard ◽  
L. Role ◽  
S. A. Siegelbaum
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Substance P ◽  
Dose Response ◽  
Calcium Current ◽  
Response Curve ◽  
Dose Response Curve ◽  
Dependent Manner ◽  
Kinase C ◽  
Voltage Dependent

1. The whole cell patch clamp was used to measure calcium current in isolated chick sympathetic ganglion neurons. Previous results showed that somatostatin inhibits calcium currents (ICa) in a voltage-dependent manner. The effect of somatostatin rapidly desensitizes. In addition, the action of somatostatin on the calcium current is inhibited by activation of protein kinase C (PKC). Because substance P (SP) has been shown to activate PKC in the chick sympathetic neurons, we here test the effects of SP on the calcium current and on the modulatory action of somatostatin. 2. At a concentration of 1 microM, SP had small, variable effects on ICa. 3. SP in the presence of guanosine 5'-triphosphate-gamma-S, or at higher concentrations (10 microM), inhibited ICa in a voltage-dependent manner, similar to the action of somatostatin. 4. Rather than inhibiting the action of somatostatin, SP (1 microM) potentiated the response to somatostatin. This effect of SP was only observed after the response to somatostatin had partially desensitized. SP had no effect on nondesensitized responses to somatostatin. 5. Desensitization of the somatostatin response involved a shift in its dose-response curve toward higher somatostatin concentrations as well as a decrease in the maximal response. SP appears to counteract the shift of the dose-response curve selectively. 6. The potentiation of the somatostatin response by SP is blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), but not by Calphostin C, Compound 5, k252a, protein kinase C (PKC)19-36, or adenylyl-imidodiphosphate (AMP-PNP), suggesting that phosphorylation is not involved and that the H-7 action does not depend on kinase inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals PDGF induction of alpha 2 integrin gene expression is mediated by protein kinase C-zeta.

1996 ◽  
Vol 134 (5) ◽  
pp. 1301-1311 ◽  
Author(s):  
J Xu ◽  
M M Zutter ◽  
S A Santoro ◽  
R A Clark
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Dermal Fibroblasts ◽  
Upstream Region ◽  
Mrna Levels ◽  
Collagen Gels ◽  
Kinase C ◽  
Pkc Zeta ◽  
Integrin Gene

Platelet-derived growth factor (PDGF) stimulates fibroblasts to move over collagen and contract three-dimensional collagen gels, processes important in wound repair and fibrocontractive diseases. These processes depend on alpha 2 beta 1 integrin ligation of collagen and PDGF induces the expression of this integrin. Several lines of evidence presented here suggest that PKC-zeta plays a role in alpha 2 integrin gene expression. The induction was blocked by chemical inhibitors for protein tyrosine kinases (PTK), genistein, and protein kinase C (PKC), chelerythrine, and bisindolylmaleimide GF 109203X. Cells depleted of phorbol 12-myristate 13-acetate (PMA)-inducible PKCs by chronic treatment with PMA still demonstrated an alpha 2 response to PDGF indicating that a non-PMA-sensitive PKC isoform was required. PDGF induced kinase activity in PKC-zeta immunoprecipitates. Antisense oligonucleotides complementary to 5' end of PKC-zeta mRNA sequences blocked the PDGF-induced increase of alpha 2 mRNA levels up to 70%, indicating PKC-zeta, a non-PMA-sensitive PKC isoform, is a component of the PDGF stimulatory pathway for alpha 2 mRNA synthesis. A 961-base pair (bp) upstream region of alpha 2 gene/CAT construct transfected into human dermal fibroblasts was positively regulated by PDGF as judged by CAT enzymatic levels. Both PTK and PKC inhibitors blocked PDGF-stimulation of the alpha 2 promoter fragment/CAT construct, indicating that the phosphorylation requirement occurred at alpha 2 promoter-directed transcription level. Therefore, we propose that PDGF-stimulatory pathway of alpha 2 integrin gene expression involves multiple cellular protein kinases, one of which is PKC-zeta.

scholarly journals Protein kinase C epsilon delays latency until anoxic depolarization through arc expression and GluR2 internalization

2017 ◽  
Vol 37 (12) ◽  
pp. 3774-3788 ◽  
Author(s):  
Charles H Cohan ◽  
Holly M Stradecki-Cohan ◽  
Kahlilia C Morris-Blanco ◽  
Nathalie Khoury ◽  
Kevin B Koronowski ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Learning And Memory ◽  
Global Cerebral Ischemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Kinase C ◽  
Anoxic Depolarization ◽  
Protein Kinase C Epsilon

Global cerebral ischemia is a debilitating injury that damages the CA1 region of the hippocampus, an area important for learning and memory. Protein kinase C epsilon (PKCɛ) activation is a critical component of many neuroprotective treatments. The ability of PKCɛ activation to regulate AMPA receptors (AMPARs) remains unexplored despite the role of AMPARs in excitotoxicity after brain ischemia. We determined that PKCɛ activation increased expression of a protein linked to learning and memory, activity-regulated cytoskeleton-associated protein (arc). Also, arc is necessary for neuroprotection and confers protection through decreasing AMPAR currents via GluR2 internalization. In vivo, activation of PKCɛ increased arc expression through a BDNF/TrkB pathway, and decreased GluR2 mRNA levels. In hippocampal cultured slices, PKCɛ activation decreased AMPAR current amplitudes in an arc- and GluR2-dependent manner. Additionally, PKCɛ activation triggered an arc- and GluR2 internalization-dependent delay in latency until anoxic depolarization. Inhibiting arc also blocked PKCɛ-mediated neuroprotection against lethal oxygen and glucose deprivation. These data characterize a novel PKCɛ-dependent mechanism that for the first time defines a role for arc and AMPAR internalization in conferring neuroprotection.

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: An effect mediated by calmodulin

2000 ◽  
Vol 78 (6) ◽  
pp. 715-723 ◽  
Author(s):  
John P Williams ◽  
Margaret A McKenna ◽  
Allyn M Thames III ◽  
Jay M McDonald
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Bone Resorption ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Fold Increase ◽  
Dependent Manner ◽  
Osteoclast Activity ◽  
Kinase C ◽  
Protein Kinase Cα

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1–0.3 µM). In contrast, tamoxifen inhibited osteoclast activity ~60% with an IC50 of 1.5 µM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase Cα recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase Cα, β, δ, ε, and ζ were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 ± 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.Key words: osteoclast, calmodulin, tamoxifen, osteoporosis, protein kinase C.

α1-Adrenoceptor subtype activation increases proto-oncogene mRNA levels. Role of protein kinase C

1998 ◽  
Vol 342 (2-3) ◽  
pp. 311-317 ◽  
Author(s):  
J.Adolfo Garcı́a-Sáinz ◽  
Rocı́o Alcántara-Hernández ◽  
José Vázquez-Prado
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Mrna Levels ◽  
Kinase C
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