PKC Signaling Pathways Research Articles

Abstract LB-365: Regulation of VEGFR-2 by leptin-Notch signaling crosstalk in mammary cancer cells

Abstract VEGFR-2 (Flk-1 or KDR) is generally recognized as the major form that mediates VEGF-induced response and the earliest marker for endothelial cell development. More importantly, VEGFR-2 directly regulates tumor angiogenesis and cancer cell survival through autocrine/paracrine mechanisms. We have previously reported that leptin signaling induced the growth of breast cancer mainly through the up-regulation of pro-angiogenic and pro-proliferative molecules, i.e., VEGF/VEGFR-2. Disruption of leptin signaling markedly reduced the growth of tumors and the expression of VEGFR-2 in mouse models of syngeneic and human breast cancer xenografts (ER+ and ER-). On the other hand, Notch signaling is essential for tumor angiogenesis. We hypothesized that a crosstalk between Notch and leptin signaling could impact the expression of VEGFR-2 in breast cancer. To test this hypothesis, the 5′-end transcription region of mouse VEGFR-2 gene was cloned and used to establish VEGFR2-Luc assays in mouse mammary tumor (MT) cells. Leptin induced VEGFR-2-Luc activity through JAK2, JNK, p38 MAP kinase, as well as PKC signaling pathways and up-regulated several members of Notch family (Notch 1∼4) in MT. Interestingly, leptin-induced VEGFR-2 transcriptional activity was significantly inhibited by a gamma-secretase inhibitor which blocks Notch pathway activation. These results suggest that leptin and Notch signaling crosstalk could impact on angiogenesis and growth of MT. Furthermore, VEGFR-2 transcription and expression was heavily depending on VEGFR-2 gene methylation and histone acetylation that could be linked to leptin's effects. Our results provide novel evidence on how VEGFR-2 could be regulated in breast cancer. Present data reinforce the idea that disruption of leptin signaling could reduce tumor angiogenesis/growth by inhibiting leptin-mediated up-regulation of VEGFR-2 and/or by negatively impacting on signals from some key members of Notch family. This might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis. [This work was supported in part by NIH/NCI 1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1; NIH/UAB Breast SPORE Career Development Award and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP)]. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-365.

Activating ERK Signaling enhances 1alpha,25(OH)2D3‐regulated 25‐hydroxyvitamin D‐24‐hydroxylase (CYP24) gene expression through the transcription factor Sp3 pathway in Caco‐2 cells.

CYP24 is an enzyme critical for degradation of 1, 25 dihydroxyvitamin (1,25D) and its gene is strongly regulated by 1,25D. Multiple signaling pathways have been shown to modulate 1,25D‐mediated CYP24 gene expression. Phorbol ester (PMA) treatment activates PKC, as well as ERK1/2 and p38 MAP kinase signaling pathways. In addition, PMA treatment enhances 1,25D‐induced CYP24 gene transcription (mRNA, 180%; reporter gene 150–250%) in Caco‐2 cells. Inhibition of PKC, ERK, and p38 kinase signaling pathways each reduced PMA enhanced and 1,25D induced CYP24 mRNA and reporter genes by 20–40%. Mithramycin A, an inhibitor of Sp protein binding to DNA, dose‐dependently reduced both 1,25D‐induced and PMA‐enhanced CYP24 promoter activity. Bioinformatics revealed 11 putative Sp1/Sp3 sites in the −298 to +74 bp region of the human CYP24 promoter. siRNA knockout of Sp1 reduced 1,25D action by only 30% but had no effect on PMA‐mediated enhancement of CYP24 induction. In contrast, Sp3 siRNA decreased both 1,25D‐mediated CYP24 induction and PMA enhancement of that effect by 20%. Immunoprecipitation showed that PMA can phosphorylate a serine residue on Sp3 and that this is blocked by inhibition of MEK1 with U0126. These data show that a portion of the impact that PMA has on 1,25D‐mediated CYP24 induction is due to activation of ERK1/2 and subsequent phosphorylation/activation of the Sp3 transcription factor. Supported by NIDDK Award DK054111 to JCF.Grant Funding Source: NIDDK Award DK054111 to JCF

Exposure to lanthanum compound diminishes LPS-induced inflammation-associated gene expression: involvements of PKC and NF-κB signaling pathways

Lanthanum chloride, a rare earth compound, possesses antibacterial and cellular immunity regulating properties. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the effects of lanthanum chloride on the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), the expression of inducible NO synthase (iNOS) and TNF-alpha in RAW 264.7 cells, a mouse macrophage cell line. We found that the LPS-elicited excessive production of NO and TNF-alpha in RAW 264.7 cells was inhibited significantly in the presence of lanthanum chloride, and the attenuation of iNOS and TNF-alpha occurred at mRNA level. Furthermore, the possible signaling components affected by lanthanum chloride in the pathway that lead to LPS-induced iNOS and TNF-alpha expression were explored. The results indicated the involvements of PKC/Ca(2+) and NF-kappaB in the attenuation of NO and pro-inflammatory cytokine production by lanthanum chloride. Our observations suggest a possible therapeutic application of this agent for treating inflammatory diseases.

PPARγ agonists upregulate the barrier function of tight junctions via a PKC pathway in human nasal epithelial cells

Peroxisome proliferator activated (PPAR)γ plays a critical role in the control of not only adipocyte differentiation, lipid metabolism and immunity but also the barrier functions of epithelial and endothelial cells. In the present study, to investigate effects of PPARγ agonists on the tight junctional barrier of human nasal epithelial cells (HNECs), hTERT-transfected HNECs, which highly express both PPARγ and tight junction proteins, were treated with the PPARγ agonists rosiglitazone and troglitazone. Treatment with the PPARγ agonists enhanced the barrier function of hTERT-transfected HNECs together with the upregulation of tight junction molecules claudin-1 and -4, occludin, and tricellulin at the transcriptional level. A significant increase of tight junction strands was also observed after treatment with rosiglitazone. Treatment with PPARγ agonists induced the activity of phospho-PKC in hTERT-transfected HNECs. The upregulation of the tight junction molecules in hTERT-transfected HNECs by rosiglitazone was inhibited by not only PPARγ antagonists GW9662 and T0070907, but also the panPKC inhibitor GF109203X.These findings suggest that PPARγ agonists upregulate the barrier function of tight junctions of human nasal epithelial cells via a PKC signaling pathway and could be novel drugs for protection against inhaled substances and pathogens in the airway epithelium of human nasal mucosa.

Vav1 couples the T cell receptor to cAMP response element activation via a PKC-dependent pathway

The transcription factor cAMP-responsive element binding protein (CREB) is a regulator of the expression of several genes important for lymphocyte activation and proliferation. However, the proximal signaling events leading to activation of CREB in T cells upon antigen receptor stimulation remain unknown. Here we identify a role for Vav1 in the activation of the cAMP response element (CRE), the binding site for CREB. T cell receptor (TCR)/CD28 — induced costimulation of Jurkat T cells expressing Vav1 but not a GEF-deficient mutant showed increased CRE activation (7.2 ± 2.4 fold over control), whereas Vav1 downregulation by siRNA reduced activation of CRE by 2.6 ± 1.3 fold. Inhibition of PKC and MEK but not p38 could reduce Vav1-mediated CRE activation, suggesting that Vav1 transmits TCR and CD28 signals to activation of CRE via PKC and ERK signaling pathways. As a consequence, downregulation of Vav1 impaired the expression of several CRE-containing genes like cyclin D1, INFγ and IL-2, whereas overexpression of Vav1 enhanced CRE-dependent gene expression. Furthermore, cAMP-induced CRE-dependent transcription and gene expression was also modulated by Vav1, but did not require activation of PKC and the GEF function of Vav1. Our data provide insights into the signal transduction events regulating CRE-mediated gene expression in T cells, which affects T cell development, proliferation and activation. We identify Vav1 as an essential component of TCR-induced CRE activation and gene expression, which underlines the central role for Vav1 as key player for TCR signal transduction and gene expression.

Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome‐specific and proteolytically released protein

Melanosomes are organelles specialized for the production of melanin pigment and are specifically produced by melanocytic cells. More than 150 pigmentation-related genes have been identified, including glycoprotein nonmetastatic melanoma protein b (GPNMB). A recent proteomics analysis revealed that GPNMB is localized in melanosomes, and GPNMB is a membrane-bound glycoprotein that shows high homology with a well-known melanosomal structural protein, Pmel17/gp100. In this study, we show that GPNMB is expressed in melanocytes of normal human skin, as well as in human melanoma cells. GPNMB is heavily glycosylated and is enriched in mature (stage III and IV) melanosomes in contrast to MART-1 and Pmel17, which are abundant in early (stage I and II) melanosomes. MART-1 and Pmel17 play critical roles in the maturation of early melanosomes; thus, we speculate that GPNMB might be important in the functions of late melanosomes, possibly their transport and/or transfer to keratinocytes. We also demonstrate that a secreted form of GPNMB is released by ectodomain shedding from the largely Golgi-modified form of GPNMB and that the PKC and Ca(2+) intracellular signaling pathways regulate that shedding. We conclude that GPNMB is a melanosomal protein that is released by proteolytic ectodomain shedding and might be a useful and specific histological marker of melanocytic cells.

Signaling and biological effects of glucagon-like peptide 1 on the differentiation of mesenchymal stem cells from human bone marrow

Glucagon-like peptide 1 (GLP-1) functions as an incretin hormone with antidiabetogenic properties. However, the role of GLP-1 in human bone marrow-derived mesenchymal stem cells (hMSCs), if any, remains unknown. The effects of GLP-1 on hMSCs were tested with regard to cell proliferation, cytoprotection, and cell differentiation into adipocytes. The signaling pathways involved in these processes were also analyzed. Cells were characterized with biochemical and morphological approaches before and after being induced to differentiate into adipocytes. PCNA protein levels were used as a proliferation index, whereas cell apoptosis was studied by deprivation of fetal bovine serum. Isolated hMSCs expressed stem cell markers as well as mRNA and GLP-1 receptor protein. GLP-1 increased the proliferation of hMSCs, which decreased when they were induced to differentiate into adipocytes. This process produced biochemical and morphological changes in cells expressing PPARgamma, C/EBPbeta, AP2, and LPL in a time-dependent pattern. Notably, GLP-1 significantly reduced the expression of PPARgamma, C/EBPbeta, and LPL. These effects were exerted at least through the MEK and PKC signaling pathways. In addition, GLP-1 significantly reduced cell apoptosis. Our data indicate that, in hMSCs, GLP-1 promotes cellular proliferation and cytoprotection and prevents cell differentiation into adipocytes. These latter findings underscore the potential therapeutic role of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and, additionally, could bolster the maintenance of hMSC stores by promoting the proliferation and cytoprotection of undifferentiated hMSC.

An αIIb-Mediated Outside-in Signaling Pathway That Causes Storage Granule Secretion and Thromboxane A2 Production.

Abstract 4011Poster Board III-947Bidirectional signaling is an essential feature of integrin αIIbβ3 function. Outside-in signal transduction mediated by αIIbβ3 is required for normal hemostasis, clot retraction, platelet aggregation and spreading. In our previous work (Blood. 2005; 105: 4345-52), we showed that αIIb-mediated outside-in signaling is enhanced in human platelets (designated VTGβ3Δ724) lacking the terminal 39 residues of the β3 cytoplasmic tail. This enhanced signaling was detected as thromboxane A2 (TxA2) production and granule secretion, and required ligand cross-linking of αIIbβ3 and platelet aggregation. This αIIbβ3-mediated outside-in signaling was specifically inhibited by a palmitoylated, but not a scrambled or non-palmitoylated version of a peptide corresponding to β3 cytoplasmic domain (CD) residues R724-R734. Inhibition presumably occurred because the corresponding region within the β3 CD (RKEFAKFEEER) can bind to the CD of αIIb and negatively regulate αIIb-mediated outside-in signaling. In this study, we elucidate part of the signaling pathway(s) underlying these αIIb-elicited events. The data presented here demonstrate that D3+Fg-induced TxA2 production and granule secretion by VTGβ3Δ724 platelets are Src family kinase(s) (SFK), PI3K, PKC and intracellular Ca2+ flux, but not ERK dependent. Further biochemical analysis demonstrated that D3+Fg treatment of control platelets ± 10U/ml apyrase and 75mM indomethacin, induced phosphorylation of ERK and slight phosphorylation of p38, but not Akt. ERK activation was not dependent on a SFK(S) or PI3K since 10mM SFK of the inhibitor PP2 and 100nM PI3K inhibitor wortmannin did not block phosphorylation of ERK. However, Akt, and p38 were intensely phosphorylated in VTGβ3Δ724 platelets treated with D3+Fg ± apyrase and indomethacin, meaning that activation of Akt, and p38 appears to be directly coupled to αIIb-mediated signaling, and not coupled to TxA2 and ADP-driven signaling. Phosphorylation of p38 and Akt, but not ERK, was totally blocked by 10mM PP2. Wortmannin (100nM) inhibited phosphorylation of Akt, but not P38 and ERK. Therefore, SFK-PI3K/Akt-PKC play an important role in αIIb-mediated signaling. Akt phosphorylation in VTGβ3Δ724 platelets stimulated by D3+Fg was also specifically inhibited by a palmitoylated β3R724-R734 peptide but not by a palmitoylated scrambled control peptide, which further proves that SFK-PI3K signaling pathway is mediated specifically by αIIb. The role of αIIb-mediated SFK-dependent PI3K/Akt signaling was investigated in normal washed platelets by stimulating those platelets with a level of the PAR4 peptide that caused secretion-dependent aggregation. The aggregation initiated αIIbβ3-mediated outside-in signaling in both mouse and human washed platelets. The PAR4 peptide-induced Akt phosphorylation, TxA2 production and granule secretion were αIIbβ3, aggregation, and PI3K-dependent, in the absence of ADP and TxA2 initiated signaling. The palmitoylated peptide β3R724-R734 (10mM), but not a control peptide blocked ATP secretion, TxA2 production and Akt phosphorylation in normal platelets stimulated with the PAR4 peptide. Therefore, the αIIb-initiated SFK(s), PI3K/Akt, PKC signaling pathway is responsible for TxA2 production and granule secretion, in both VTGβ3Δ724 and normal platelets. SFK-dependent p38 activation is also triggered by αIIb-mediated signaling, but it is not Akt-dependent and the function of p38 in αIIb-mediated signaling events is unknown. Finally, ERK activation is αIIbβ3-dependent, but not αIIbβ3-mediated; therefore ERK activation probably is an effect of non-αIIb-mediated contact-dependent signaling. The work presented here provides a new understanding of αIIbb3- outside-in signaling. Disclosures:No relevant conflicts of interest to declare.

Fatty acids induce amylin expression and secretion by pancreatic β-cells

Amylin is the major component of pancreatic amyloid, which is implicated in the development of type 2 diabetes. It is costored with insulin in the secretory granules of pancreatic beta-cells and cosecreted with insulin following stimulation with glucose. Here, we investigate the effect of fatty acids (FAs) on amylin expression and secretion by beta-cells and explore the underlying mechanisms. Palmitate and oleate dose-dependently induced amylin mRNA accumulation in murine pancreatic beta-cell line MIN6 and primary pancreatic islets. the inductive effect of FAs on amylin expression is independent of glucose concentration. FAs upregulated amylin expression at the transcriptional level, and FAs must be metabolized to induce amylin expression. FAs also significantly induced human amylin promoter activation. Pretreatment of MIN6 cells with Ca(2+) chelator (EGTA, BAPTA-AM) PKC inhibitor Gö-6976 or protein synthesis inhibitor cycloheximide significantly inhibited FA-induced amylin mRNA expression. Transcription factors cAMP-responsive element-binding protein, pancreatic and duodenal homeobox factor-1, and peroxisome proliferator-activated receptor were not involved in FA-induced amylin expression. Palmitate and oleate both increased amylin and insulin release from MIN6 cells and stimulated amylin expression but had no effect on insulin expression. Mice refed with Intralipid had significantly higher levels of plasma FFA, amylin, and insulin than those refed with saline. These data demonstrate that FAs differently regulate amylin and insulin expression and induce both amylin and insulin release. Ca(2+) and PKC signaling pathways and de novo-synthesized protein(s) were involved in FA-induced amylin expression. Induction of amylin production and release by FA may contribute to its biological functions under physiological conditions.

Vascular Endothelial Growth Factor (VEGF) Receptor-2 Tyrosine 1175 Signaling Controls VEGF-induced von Willebrand Factor Release from Endothelial Cells via Phospholipase C-γ1- and Protein Kinase A-dependent Pathways

There is increasing evidence that vascular endothelial growth factor (VEGF) contributes to inflammation independent of its angiogenic functions. Targeting some of the components in endothelial Weibel-Palade bodies (WPBs) effectively inhibits VEGF-induced inflammation, but little is known about how VEGF regulates WPB exocytosis. In this study, we showed that VEGF receptor-2 (VEGFR2), but not VEGFR1, is responsible for VEGF-induced release of von Willebrand factor (vWF), a major marker of WPBs. This is in good contrast to VEGF-stimulated interleukin-6 release from endothelium, which is selectively mediated through VEGFR1. We further demonstrated that VEGFR2-initiated phospholipase C-gamma1 (PLCgamma1)/calcium signaling is important but insufficient for full vWF release, suggesting the possible participation of another effector pathway. We found that cAMP/protein kinase A (PKA) signaling is required for full vWF release. Importantly, a single mutation of Tyr(1175) in the C terminus of VEGFR2, a tyrosine residue crucial for embryonic vasculogenesis, abolished vWF release, concomitant with defective activations of both PLCgamma1 and PKA. These data suggest that Tyr(1175) mediates both PLCgamma1-dependent and PKA-dependent signaling pathways. Taken together, our results not only reveal a novel Tyr(1175)-mediated signaling pathway but also highlight a potentially new therapeutic target for the management of vascular inflammation.

Evidence of TRPV1 receptor and PKC signaling pathway in the antinociceptive effect of amyrin octanoate

Previous studies from our group investigated the antinociceptive property of amyrin octanoate, a synthetic compound derivative from natural precursor α, β-amyrin, against nociceptive response induced by acetic acid and formalin. Here, we investigated some of the mechanisms of action underlying the antinociceptive effects of amyrin octanoate. Amyrin octanoate given intraperitoneally (0.001–1 mg /kg) or intrathecally (10–1000 ng /site) caused dose-dependent and long-lasting inhibition of acetic acid-induced visceral nociception, with mean ID 50 values of 0.003 (0.001–0.005) mg/kg and 122.4 (60.8–246.6) ng/site, respectively. In the capsaicin- and glutamate-induced paw licking, amyrin octanoate caused significant and dose-dependent inhibition of both nociceptive responses, with ID 50 values of 1.36 and 0.04 mg/kg, respectively. Furthermore, amyrin octanoate also reduced significantly the nociception caused by intrathecal injection of glutamate, substance P and capsaicin, with inhibitions of 36 ± 11%, 67 ± 10% and 78 ± 5%, respectively. The antinociception caused by amyrin octanoate in the acetic acid test was significantly attenuated by neonatal pretreatment of mice with capsaicin, but seems to involve mechanisms independent of G i/o protein, opioidergic, serotonergic, noradrenergic and cholinergic system, since it was not affected by pertussis toxin, naloxone, yohimbine, mecamylamine or atropine. In addition, amyrin octanoate reduced thermal and mechanical hyperalgesia induced by bradykinin and phorbol myristate acetate in rats, without affecting similar responses caused by prostaglandin E 2. Taken together, the present results shown that octanoate amyrin produces antinociceptive and antihyperalgesic effects, through an interaction with capsaicin-sensitive fibers and the inhibition of the PKC signaling pathway.

The pharmacological NFκB inhibitors BAY117082 and MG132 induce cell arrest and apoptosis in leukemia cells through ROS-mitochondria pathway activation

A growing body of evidence suggests the inhibition of NFκB as a strategy to induce cell death in tumor cells. In this work, we evaluated the effects of the pharmacological NFκB inhibitors BAY117082 and MG132 on leukemia cells apoptosis. BAY117082 and MG132 presented potent apoptotic effects compared to inhibitors of MAPKs, EGFR, PI3K/Akt, PKC and PKA signaling pathways. Non-tumor peripheral blood cells were insensitive to BAY117082 and MG132 apoptotic effects. BAY117082 and MG132-induced apoptosis was dependent on their ability to increase ROS as a prelude to mitochondria membrane potential (MMP) depolarization, permeability transition pore opening and cytochrome c release. Antioxidants blocked MG132 and BAY117082 effects on ROS, MMP and cell death. Although apoptotic markers as phosphatidylserine externalization, chromatin condensation and sub-G1 were detected in BAY117082-treated cells, caspases activation did not occur and apoptosis was insensitive to caspase inhibitors, suggesting a caspase-independent mechanism. In contrast, MG132 induced classical apoptosis through ROS-mitochondria and subsequent caspase-9/caspase-3 activation. At sub-apoptotic concentrations, BAY117082 and MG132 arrested cells in G2/M phase of the cell cycle and blocked doxorubicin-induced NFκB, which sensitized doxorubicin-resistant cells. Data suggest that the NFκB inhibitors MG132 and BAY117082 are potential anti-leukemia agents.

Potentiation of spinal NMDA-mediated nociception by cocaine- and amphetamine-regulated transcript peptide via PKA and PKC signaling pathways in rats

Our previous study showed that cocaine- and amphetamine-regulated transcript peptide fragment 55–102 (CARTp) specifically potentiated spinal N-methyl- d-aspartate (NMDA)-mediated nociceptive transmission in vivo and in vitro. The cellular mechanisms underlying CARTp potentiation of NMDA receptor function remains unclear. The present study was carried out to test the hypothesis that CARTp changes the phosphorylated state of NMDA receptors by activating intracellular signals and subsequently increasing the function of NMDA receptors. We found that the potentiating effect of CARTp on spinal NMDA-induced hyperalgesia in rats was reduced by intrathecal pretreatment with KT5720 (a selective PKA inhibitor) or GF109206X (a selective PKC inhibitor), but was increased by pretreatment with calyculin A (a protein phosphatase inhibitor). In the in vitro electrophysiological study, CARTp potentiation of NMDA-induced depolarizations was blocked by superfusion of PKA or PKC inhibitor applied 10 min before the application of CARTp. The levels of phosphoserine 897 on the NR1 subunit (pNR1-ser897) and phosphoserine 896 on the NR1 subunit (pNR1-ser896) in the dorsal horn of spinal lumbar segments significantly increased following CARTp superfusion in vitro or intrathecal injection in vivo. The increases in pNR1-ser897 and pNR1-ser896 in the in vivo and in vitro studies were inhibited by pretreatment with KT5720 and GF109206X, respectively. The results provide the first evidence that CARTp increases the phosphorylation of NMDA receptor NR1 subunit via activation of PKA and PKC signals, which may play a crucial role in CARTp regulation of spinal NMDA receptor-mediated nociceptive responses.

Protein kinase C epsilon is involved in ionizing radiation induced bystander response in human cells

Our earlier study demonstrated the induction of PKC isoforms (βII, PKC-α/β, PKC-θ) by ionizing radiation induced bystander response in human cells. In this study, we extended our investigation to yet another important member of PKC family, PKC epsilon (PKCɛ). PKCɛ functions both as an anti-apoptotic and pro-apoptotic protein and it is the only PKC isozyme implicated in oncogenesis. Given the importance of PKCɛ in oncogenesis, we wished to determine whether or not PKCɛ is involved in bystander response. Gene expression array analysis demonstrated a 2–3-fold increase in PKCɛ expression in the bystander human primary fibroblast cells that were co-cultured in double-sided Mylar dishes for 3 h with human primary fibroblast cells irradiated with 5 Gy of α-particles. The elevated PKCɛ expression in bystander cells was verified by quantitative real time PCR. Suppression of PKCɛ expression by small molecule inhibitor Bisindolylmaleimide IX (Ro 31-8220) considerably reduced the frequency of micronuclei (MN) induced both by 5 Gy of γ-rays (low LET) and α-particles (high LET) in bystander cells. Similar cytoprotective effects were observed in bystander cells after siRNA mediated silencing of PKCɛ suggestive of its critical role in mediating some of the bystander effects (BE). Our novel study suggests the possibility that PKC signaling pathway may be a critical molecular target for suppression of ionizing radiation induced biological effects in bystander cells.

Chronic hypoxia increases pressure-dependent myogenic tone of the uterine artery in pregnant sheep: role of ERK/PKC pathway

Chronic hypoxia during pregnancy has profound effects on uterine artery (UA) contractility and attenuates uterine blood flow. The present study tested the hypothesis that chronic hypoxia inhibits the pregnancy-induced reduction in pressure-dependent myogenic tone of resistance-sized UAs. UAs were isolated from nonpregnant ewes (NPUAs) and near-term pregnant ewes (PUAs) that had been maintained at sea level (approximately 300 m) or at high altitude (3,801 m) for 110 days. In normoxic animals, the pressure-dependent myogenic response was significantly attenuated in PUAs compared with NPUAs. Hypoxia significantly increased myogenic tone in PUAs and abolished its difference between PUAs and NPUAs. Consistently, there was a significant increase in PKC-mediated baseline Ca(2+) sensitivity of PUAs in hypoxic animals. Hypoxia significantly increased phorbol 12,13-dibutyrate (PDBu)-induced contractions in PUAs but not in NPUAs. Whereas the inhibition of ERK1/2 by PD-98059 potentiated PDBu-mediated contractions of PUAs in normoxic animals, it failed to do so in hypoxic animals. Hypoxia decreased ERK1/2 expression in PUAs. PDBu induced membrane translocation of PKC-alpha and PKC-epsilon. Whereas there were no significant differences in PKC-alpha translocation among all groups, the translocation of PKC-epsilon was significantly enhanced in NPUAs compared with PUAs in normoxic animals, and hypoxia significantly increased PKC-epsilon translocation in PUAs. In the presence of PD-98059, there were no significant differences in PDBu-induced PKC-epsilon translocation among all groups. Treatment of PUAs isolated from normoxic animals with 10.5% O(2) for 48 h ex vivo significantly increased PDBu-induced contractions and eliminated its difference between PUAs and NPUAs. The results suggest that hypoxia upregulates pressure-dependent myogenic tone through its direct effect in suppressing ERK1/2 activity and increasing the PKC signal pathway, leading to an increase in the Ca(2+) sensitivity of the myogenic mechanism in the UA during pregnancy.

Regulation of swelling-activated Cl channel in HEK 293 cells by extracellular low pH

Using voltage-clamped HEK 293 cells that were exposed to hypotonic solution, we measured the effects of low pH on the slowly (∼100s) swelling-activated Cl-current. The channel showed mild outward rectification during ramp-clamps, had a reversal potential (-21.7 ± 2.9 mV) close to the predicted ECl (-19.1mV), and was reversibly inhibited by DIDS. Changing extracellular pH from 7.4 to 6.0 significantly reduced the current and accelerated its inactivation measured over 200 ms at +80 mV: In cells with minimal Ca2+-buffers (0.1mM EGTA), challenging with hypotonic solution at pH 6.0 reduced the initial and final currents by 49% and 55%, respectively (compared to pH 7.4 control values). Interestingly, in highly Ca2+-buffered cells (10 mM BAPTA), the decay of the current at pH 6.0 was significantly faster with 49.1 % initial and 74.7% final suppression.We also found that the current was reduced by 75% by 5 μM U-73122 (an inhibitor of phospholipase C) and by 30% by 20 μM Farnesyl thiotriazole (a PKC activator). High intracellular Mg2+ (10.7 mM) nearly abolished activation of the current suppressing its slope conductance from 7.0 ± 0.2 to 2.1 ± 0.3 nS at +80 mV and from 4.4 ± 0.2 to 0.33 ± 0.1 nS at -80 mV (p<0.001). Extracellular Mg2+ (10 mM) had no significant effect on the current. Intracellular cAMP (200 μm) delayed, but did not prevent, the activation of the current. Extracellular cAMP suppressed 75% of the current.These data suggest that the kinetics of the inactivation of the proton-regulated chloride channel depend on the intracellular buffering capacity for Ca2+ and that the magnitude of the current is regulated by PIP2, PKC, and cAMP signaling pathways and by intracellular Mg2+.

A role for Syndecan-4 in neural induction involving ERK- and PKC-dependent pathways

Syndecan-4 (Syn4) is a heparan sulphate proteoglycan that is able to bind to some growth factors, including FGF, and can control cell migration. Here we describe a new role for Syn4 in neural induction in Xenopus. Syn4 is expressed in dorsal ectoderm and becomes restricted to the neural plate. Knockdown with antisense morpholino oligonucleotides reveals that Syn4 is required for the expression of neural markers in the neural plate and in neuralised animal caps. Injection of Syn4 mRNA induces the cell-autonomous expression of neural, but not mesodermal, markers. We show that two parallel pathways are involved in the neuralising activity of Syn4: FGF/ERK, which is sensitive to dominant-negative FGF receptor and to the inhibitors SU5402 and U0126, and a PKC pathway, which is dependent on the intracellular domain of Syn4. Neural induction by Syn4 through the PKC pathway requires inhibition of PKCdelta and activation of PKCalpha. We show that PKCalpha inhibits Rac GTPase and that c-Jun is a target of Rac. These findings might account for previous reports implicating PKC in neural induction and allow us to propose a link between FGF and PKC signalling pathways during neural induction.

Wnt5a Is Required for Endothelial Differentiation of Embryonic Stem Cells and Vascularization via Pathways Involving Both Wnt/β-Catenin and Protein Kinase Cα

In this study, we examined the signaling pathways activated by Wnt5a in endothelial differentiation of embryonic stem (ES) cells and the function of Wnt5a during vascular development. We first found that Wnt5a(-/-) mouse embryonic stem (mES) cells exhibited a defect in endothelial differentiation, which was rescued by addition of Wnt5a, suggesting that Wnt5a is required for endothelial differentiation of ES cells. Involvement of both beta-catenin and protein kinase (PK)Calpha pathways in endothelial differentiation of mES cells requiring Wnt5a was indicated by activation of both beta-catenin and PKCalpha in Wnt5a(+/-) but not in Wnt5a(-/-) mES cells. We also found that beta-catenin or PKCalpha knockdowns inhibited the Wnt5a-induced endothelial differentiation of ES cells. Moreover, the lack of endothelial differentiation of Wnt5a(-/-) mES cells was rescued only by transfection of both beta-catenin and PKCalpha, indicating that both genes are required for Wnt5a-mediated endothelial differentiation. Wnt5a was also found to be essential for the differentiation of mES cells into immature endothelial progenitor cells, which are known to play a role in repair of damaged endothelium. Furthermore, a defect in the vascularization of the neural tissue was detected at embryonic day 14.5 in Wnt5a(-/-) mice, implicating Wnt5a in vascular development in vivo. Thus, we conclude that Wnt5a is involved in the endothelial differentiation of ES cells via both Wnt/beta-catenin and PKC signaling pathways and regulates embryonic vascular development.

Effect of curcumin on acidic pH-induced expression of IL-6 and IL-8 in human esophageal epithelial cells (HET-1A): role of PKC, MAPKs, and NF-κB

Human esophageal epithelial cells play a key role in esophageal inflammation in response to acidic pH during gastroesophageal reflux disease (GERD), increasing secretion of IL-6 and IL-8. The mechanisms underlying IL-6 and IL-8 expression and secretion in esophageal epithelial cells after acid stimulation are not well characterized. We investigated the role of PKC, MAPK, and NF-kappaB signaling pathways and transcriptional regulation of IL-6 and IL-8 expression in HET-1A cells exposed to acid. Exposure of HET-1A cells to pH 4.5 induced NF-kappaB activity and enhanced IL-6 and IL-8 secretion and mRNA and protein expression. Acid stimulation of HET-1A cells also resulted in activation of MAPKs and PKC (alpha and epsilon). Curcumin, as well as inhibitors of NF-kappaB (SN-50), PKC (chelerythrine), and p44/42 MAPK (PD-098059) abolished the acid-induced expression of IL-6 and IL-8. The JNK inhibitor SP-600125 blocked expression/secretion of IL-6 but only partially attenuated IL-8 expression. The p38 MAPK inhibitor SB-203580 did not inhibit IL-6 expression but exerted a stronger inhibitory effect on IL-8 expression. Together, these data demonstrate that 1) acid is a potent inducer of IL-6 and IL-8 production in HET-1A cells; 2) MAPK and PKC signaling play a key regulatory role in acid-mediated IL-6 and IL-8 expression via NF-kappaB activation; and 3) the anti-inflammatory plant compound curcumin inhibits esophageal activation in response to acid. Thus IL-6 and IL-8 expression by acid may contribute to the pathobiology of mucosal injury in GERD, and inhibition of the NF-kappaB/proinflammatory cytokine pathways may emerge as important therapeutic targets for treatment of esophageal inflammation.

Ethambutol induces PKC-dependent cytotoxic and antiproliferative effects on human retinal pigment cells

Ethambutol (EMB)-induced ocular side effects may involve the influence on functions of retinal pigment epithelium (RPE), in addition to EMB-induced optic neuropathy. To address this issue, the molecular and cellular effects of EMB on RPE including growth regulation, morphological responses, phagocytic activity, and the relevant signaling pathways were investigated. EMB (at optimal concentration 8.0 mM) can trigger cell cycle arrest in both RPE50 and ARPE19 cells, accompanied by reduced DNA synthesis. EMB also induced cytoplasmic vacuole formation in both RPE cell lines. Under transmission electric microscope, the phagosomes were replaced by vacuoles and the number of microvilli was reduced in EMB-treated cells. Animal experiments also demonstrated the vacuole formation within RPE of the EMB-treated rats. On the other hand, by in vitro phagocytosis assay using rod outer segment (ROS) as the target, we found EMB suppressed phagocytosis in the cultured RPE, which is consistent with the decreased rhodopsin uptake in the RPE of the EMB-treated rats. Furthermore, inhibitor of protein kinase C but not MAPK, prevented the EMB-induced phenotypical changes. Using a non-radioactive PKC assay, we also demonstrated the PKC activity in both RPE cell lines can be induced by EMB. In conclusion, EMB may exert toxic effects in RPE including suppression of cell growth, formation of cytoplasmic vacuoles and reduction of phagocytic functions via PKC signal pathway.