scholarly journals Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β

2019 ◽  
Vol 294 (43) ◽  
pp. 15759-15767 ◽  
Author(s):  
Ying Wang ◽  
Luke H. Hoeppner ◽  
Ramcharan Singh Angom ◽  
Enfeng Wang ◽  
Shamit Dutta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase D ◽  
Vegf Receptor

scholarly journals Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells

2010 ◽  
Vol 429 (3) ◽  
pp. 565-572 ◽  
Author(s):  
Ian M. Evans ◽  
Azadeh Bagherzadeh ◽  
Mark Charles ◽  
Tony Raynham ◽  
Chris Ireson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Umbilical Vein ◽  
Biological Effects ◽  
Camp Response Element Binding ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase D ◽  
Vegf Receptor

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.

scholarly journals Protein Kinase D Mediates Mitochondrion-to-Nucleus Signaling and Detoxification from Mitochondrial Reactive Oxygen Species

2005 ◽  
Vol 25 (19) ◽  
pp. 8520-8530 ◽  
Author(s):  
Peter Storz ◽  
Heike Döppler ◽  
Alex Toker
Keyword(s):  
Reactive Oxygen Species ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Reactive Oxygen ◽  
Protein Kinase D ◽  
Oxygen Species ◽  
Mitochondrial Reactive Oxygen Species ◽  
Mitochondrial Oxidative Stress ◽  
Cellular Survival ◽  
Mitochondrial Ros

ABSTRACT Efficient elimination of mitochondrial reactive oxygen species (mROS) correlates with increased cellular survival and organism life span. Detoxification of mitochondrial ROS is regulated by induction of the nuclear SOD2 gene, which encodes the manganese-dependent superoxide dismutase (MnSOD). However, the mechanisms by which mitochondrial oxidative stress activates cellular signaling pathways leading to induction of nuclear genes are not known. Here we demonstrate that release of mROS activates a signal relay pathway in which the serine/threonine protein kinase D (PKD) activates the NF-κB transcription factor, leading to induction of SOD2. Conversely, the FOXO3a transcription factor is dispensable for mROS-induced SOD2 induction. PKD-mediated MnSOD expression promotes increased survival of cells upon release of mROS, suggesting that mitochondrion-to-nucleus signaling is necessary for efficient detoxification mechanisms and cellular viability.

Abstract 342: The Antiapoptotic Function of APEX Nuclease (Multifunctional DNA Repair Enzyme) 1 in Endothelial Cells Is Linked to Changes in Transcription Factor Activities and Independent of Its DNA Repair Domain

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Nadine Dyballa-Rukes ◽  
Lothar Rabanter ◽  
Christine Goy ◽  
Joachim Altschmied ◽  
Judith Haendeler
Keyword(s):  
Endothelial Cells ◽  
Dna Repair ◽  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Redox Activity ◽  
Nuclear Staining ◽  
Repair Enzyme ◽  
H2o2 Treatment ◽  
The Impact

APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has both DNA repair and redox regulatory activity. Interestingly, APEX1 is also able to modulate transcription factor reduction, a prerequisite for DNA binding, independent of its intrinsic redox activity, probably by recruiting other reducing molecules like Thioredoxin-1 (TXN). APEX1 has been shown to exert anti-apoptotic properties in different cell lines and is localized in the cytosol and in the nucleus. So far it has not been studied whether APEX1 acts anti-apoptotic in primary human endothelial cells (EC) and whether this is dependent on its nuclear or cytosolic localization. After cloning human APEX1 and overexpression in EC we demonstrated that it inhibits not only basal but also H2O2-induced apoptosis. To further characterize the anti-apoptotic properties of APEX1 we generated mutants that lacked the C-terminal DNA repair domain (APEX1ΔC 1-127) or the N-terminal nuclear localization signal (NLS) (APEX1ΔN 21-318). We first analyzed the localization of the mutants by immunostaining. APEX1wt and APEX1ΔC 1-127 showed a predominant nuclear staining whereas APEX1ΔN 21-318 was mainly localized in the cytosol. With respect to the anti-apoptotic properties, only the mutant with intact NLS and redox domain APEX1ΔC 1-127 prevented apoptosis both under basal conditions as well as after H2O2 treatment similar to APEX1wt. The association between nuclear localization of APEX1wt and its anti-apoptotic properties in EC suggests that this function is linked to changes in transcription factor activities. It is known that the p65 subunit of the transcription factor complex NF-kB is activated by pro-apoptotic stimuli in EC. Using a specific DNA binding assay we could show that overexpression of APEX1wt and APEX1ΔC 1-127 reduced DNA binding of p65, whereas the NLS-deficient mutant had no effect. In conclusion, the anti-apoptotic activity of APEX1 in EC critically depends on its nuclear localization and redox-activity, is independent of its DNA repair domain and is at least partially due to suppression of NF-kB dependent, pro-apoptotic gene expression programs. In future studies we will analyze the impact of APEX1 on other transcription factors in concert with TXN.

scholarly journals TCF21 inhibits tumor-associated angiogenesis and suppresses the growth of cholangiocarcinoma by targeting PI3K/Akt and ERK signaling

2019 ◽  
Vol 316 (6) ◽  
pp. G763-G773 ◽  
Author(s):  
Hua-Xin Duan ◽  
Bo-Wen Li ◽  
Xin Zhuang ◽  
Lu-Ting Wang ◽  
Qian Cao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Cell Lines ◽  
Protein Kinase B ◽  
Tube Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Xenograft Growth

Tumor-associated angiogenesis plays a critical role in the pathogenesis of cholangiocarcinoma (CCA). In this study, we examined the biological effects and molecular mechanisms of transcription factor 21 (TCF21) on CCA-associated angiogenesis. TCF21 expression was compared between 15 pairs of peritumor normal tissues and CCA tissues and also between normal bile duct epithelial cells and two CCA cell lines (QBC-939 and TFK-1) using real-time PCR and Western blot. With the use of both CCA cell lines as the model system, we stably expressed TCF21 by lentiviral transduction (Lv-TCF21). In vivo, we monitored xenograft growth from different CCA cells, measured tumor-associated angiogenesis by histological analysis, and determined the expressions and circulatory levels of VEGFA and PDGF-BB by immunohistochemistry and ELISA, respectively. In vitro, we assessed the effects of conditioned medium collected from different CCA cells on the viability, migration, and tube formation of endothelial cells and explored the significance of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), as well as ERK1/2 signaling in this process. TCF21 was significantly downregulated in CCA tissues or cell lines. Ectopic expression of TCF21 in CCA cells inhibited xenograft growth or tumor-associated angiogenesis in vivo and targeted the expression and secretion of proangiogenic factors, VEGFA and PDGF-BB. In vitro, the conditioned medium collected from Lv-TCF21 CCA cells significantly reduced the viability, migration, and tube formation of endothelial cells. On the molecular level, the targeting of PI3K/Akt and ERK1/2 signaling mediated the anti-angiogenic activity of TCF21. TCF21 presents growth-inhibitory and anti-angiogenic activities, and thus the elevation of TCF21 expression may provide therapeutic benefits for CCA. NEW & NOTEWORTHY Transcription factor 21 (TCF21) is downregulated in cholangiocarcinoma (CCA) tissues or cells. TCF21 inhibits the growth of xenografts derived from CCA cells. TCF21 suppresses in vivo tumor-associated angiogenesis. TCF21 targets expression and production of proangiogenic factors from CCA cells. The targeting of phosphatidylinositol 3-kinase/protein kinase B and ERK1/2 signaling mediates the anti-angiogenesis of TCF21.

Shuttling of HDAC5 in H9C2 cells regulates YY1 function through CaMKIV/PKD and PP2A

2006 ◽  
Vol 291 (5) ◽  
pp. C1029-C1037 ◽  
Author(s):  
Carmen C. Sucharov ◽  
Stephen Langer ◽  
Michael Bristow ◽  
Leslie Leinwand
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Detailed Analysis ◽  
Histone Deacetylase ◽  
Cardiac Myocytes ◽  
Protein Kinase D ◽  
H9c2 Cells ◽  
Signaling Mechanism ◽  
Differentiated Cells ◽  
Phosphatase 2A

YY1 is a transcription factor that can activate or repress transcription of a variety of genes and is involved in several developmental processes. YY1 is a repressor of transcription in differentiated H9C2 cells and in neonatal cardiac myocytes but an activator of transcription in undifferentiated H9C2 cells. We now present a detailed analysis of the functional domains of YY1 when it is acting as a repressor or an activator and identify the mechanism whereby its function is regulated in the differentiation of H9C2 cells. We show that histone deacetylase 5 (HDAC5) is localized to the cytoplasm in undifferentiated H9C2 cells and that this localization is dependent on Ca2+/calmodulin-dependent kinase IV (CaMKIV) and/or protein kinase D (PKD). In differentiated cells, HDAC5 is nuclear and interacts with YY1. Finally, we show that HDAC5 localization in differentiated cells is dependent on phosphatase 2A (PP2A). Our results suggest that a signaling mechanism that involves CaMKIV/PKD and PP2A controls YY1 function through regulation of HDAC5 and is important in the maintenance of muscle differentiation.

scholarly journals Inhibition of Nuclear Import by Protein Kinase B (Akt) Regulates the Subcellular Distribution and Activity of the Forkhead Transcription Factor AFX

2001 ◽  
Vol 21 (10) ◽  
pp. 3534-3546 ◽  
Author(s):  
Amy M. Brownawell ◽  
Geert J. P. L. Kops ◽  
Ian G. Macara ◽  
Boudewijn M. T. Burgering
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Transcriptional Activity ◽  
Protein Kinase B ◽  
Phosphorylation Site ◽  
Nuclear Accumulation ◽  
Forkhead Transcription Factor ◽  
Akt Phosphorylation

ABSTRACT AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

scholarly journals Thyroid hormone induced angiogenesis through the integrin αvβ3/protein kinase D/histone deacetylase 5 signaling pathway

2014 ◽  
Vol 52 (3) ◽  
pp. 245-254 ◽  
Author(s):  
Xin Liu ◽  
Nan Zheng ◽  
Ya-Nan Shi ◽  
Jihong Yuan ◽  
Lanying Li
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Growth Factor ◽  
Thyroid Hormone ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
Histone Deacetylase ◽  
Nuclear Export ◽  
Integrin Αvβ3 ◽  
Protein Kinase D

Thyroid hormone is reported to induce angiogenesis, which is mediated by the membrane receptor integrin αvβ3, but the precise signaling pathway is still not very clear. Recently, studies have shown that protein kinase D (PKD) regulates the recycling of integrin αvβ3, which is required for cell migration. Moreover, phosphorylated PKD stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in endothelial cells. As a potent pro-angiogenic growth factor, basic fibroblast growth factor (bFGF (FGF2)) is a downstream target gene of HDAC5. Therefore, we examined the hypothesis that a novel signaling pathway through integrin αvβ3/PKD/HDAC5 might contribute to thyroxine (T4)-induced angiogenesis. We selected human umbilical vein endothelial cells (HUVECs) for treatment. Angiogenesis was assessed using wound-healing and tubulogenesis assays. Signaling molecules, including phosphorylated PKD and HDAC5, were measured by western blotting. bFGF mRNA was analyzed by real-time PCR. Our results showed that T4 (100 nmol/l) stimulated the migration and formation of tube-like structures of HUVECs, whereas tetraiodothyroacetic acid (Tetrac, 100 nmol/l) inhibited T4-induced cell migration. Importantly, T4 promoted the phosphorylation of PKD and HDAC5. These effects were inhibited respectively by Tetrac, PKC inhibitor (2.5 μmol/l) and PKD siRNA. Meanwhile, T4 could promote the cytoplasmic accumulation of phosphorylated HDAC5 in HUVECs. In addition, bFGF mRNA expression in HUVECs significantly increased within 2 h of T4 treatment, but was decreased by Tetrac. Our findings indicate that T4 increases the expression of bFGF mRNA via the integrin αvβ3/PKD/HDAC5 signaling pathway, which plays an important role in angiogenesis.

Vascular endothelial growth factor induces protein kinase D-dependent production of proinflammatory cytokines in endothelial cells

2009 ◽  
Vol 296 (4) ◽  
pp. C821-C827 ◽  
Author(s):  
Qin Hao ◽  
Linping Wang ◽  
Hua Tang
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Protein Kinase ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Protein Kinase D ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor

Emerging evidence indicates that vascular endothelial growth factor (VEGF) plays a critical role in host inflammatory responses in several disease states, including atherosclerosis, sepsis, and rheumatoid arthritis. In this study, we determined the effect of VEGF on endothelial induction of proinflammatory cytokines and investigated the responsible signal pathways. By using a cytokine antibody array that detects the end point protein products released from endothelial cells (ECs), we found that VEGF, via VEGF receptor 2 (VEGFR2), predominantly induced the production of proinflammatory cytokine interleukin (IL)-6 and CXC chemokines IL-8 and growth-related oncogene-α (GRO-α) in ECs but not in leukocytes among 36 cytokines in the array. The production of these inflammatory cytokines by VEGF was much stronger than the induction of cell adhesion molecule in ECs. We further found that the cytokine production by VEGF was essentially mediated by the Gö-6976-sensitive protein kinase D (PKD) family kinases. Importantly, the VEGF-induced production of IL-6, IL-8, and GRO-α was inhibited ∼70%, 40%, or 37% by PKD1 silencing (more than 90% knockdown) with three small interference RNAs that target different PKD1 regions. Moreover, silencing PKD2 downregulated VEGFR2 and markedly inhibited the cytokine production by VEGF in ECs. Our results indicate that VEGF, via VEGFR2-PKD1 axis, induces the production of proinflammatory cytokine IL-6, IL-8, and GRO-α in ECs but not in leukocytes, which may offer new insights into the mechanism of the proinflammatory activity of VEGF.

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