Down-regulation Of Protein Kinase Research Articles

Down-regulation of protein kinase C alpha/ezrin signals in light-induced phagocytic crisis of retinal pigment epithelium cells.

To investigate the roles of PKC-α/ezrin signals in phagocytosis crisis of retinal pigment epithelium (RPE) cells in light damage model. Light induced mice RPE injury model was established by continuously irradiating cool white light at different exposure time (0, 4, 8h light intensity: 4.18×10-6 J/cm2). In vitro, human ARPE-19 cells treated with the doses and intensity (1.57×10-6 J/cm2) of laser irradiation. Histology analysis was evaluated by hematoxylin and eosin (HE) staining. In vivo RPE phagocytosis was quantified by measuring the accumulation of photoreceptor outer segments in the sub-retinal space. In vitro RPE phagocytosis was assessed by calculating the relative fluorescence intensity of FITC-labeled microspheres in ARPE-19 cells. To further investigate the molecular mechanism, the activation of PKC-α/ezrin signal was evaluated by Western blot in vivo and in vitro. HE staining revealed that the thickness of outer nuclear layer decreased significantly after 4 and 8h light exposure. By immunostaining with rhodopsin, a significant greater accumulation of photoreceptor outer segment was noticed after light injury. In vitro, light injured RPE cells had less phagocytic activity in a dose dependent manner than that of the normal control (P<0.01). Western blot suggested the activation of PKC-α/ezrin signaling was down-regulated in a dose-dependent manner after light exposure. Our data suggest that light induced phagocytic crisis of RPE cells may result from the down-regulation of PKC-α/ezrin signaling.

The helminth product, ES-62 modulates dendritic cell responses by inducing the selective autophagolysosomal degradation of TLR-transducers, as exemplified by PKCδ.

We have previously shown that ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the parasitic filarial nematode Acanthocheilonema viteae targets dendritic cell (DC) responses, specifically by suppressing TLR4 signalling to inhibit Th1/Th17-driven inflammation. We have now investigated the molecular mechanisms underpinning such immunomodulation and show here that ES-62-mediated downregulation of protein kinase C-δ (PKC-δ), a TLR4-associated signalling mediator required for full activation of LPS-driven pro-inflammatory responses, is associated with induction of a low level of autophagic flux, as evidenced by upregulation and trafficking of p62 and LC3 and their consequent autophagolysosomal degradation. By contrast, the classical TLR4 ligand LPS, strongly upregulates p62 and LC3 expression but under such canonical TLR4 signalling this upregulation appears to reflect a block in autophagic flux, with these elements predominantly degraded in a proteasomal manner. These data are consistent with autophagic flux acting to homeostatically suppress proinflammatory DC responses and indeed, blocking of PKC-δ degradation by the autophagolysosomal inhibitors, E64d plus pepstatin A, results in abrogation of the ES-62-mediated suppression of LPS-driven release of IL-6, IL-12p70 and TNF-α by DCs. Thus, by harnessing this homeostatic regulatory mechanism, ES-62 can protect against aberrant inflammation, either to promote parasite survival or serendipitously, exhibit therapeutic potential in inflammatory disease.

Down-regulation of protein kinase, DNA-activated, catalytic polypeptide attenuates tumor progression and is an independent prognostic predictor of survival in prostate cancer

ObjectiveProtein kinase, DNA-activated, catalytic polypeptide (PRKDC) is a critical component of DNA repair machinery and its dysregulated expression has been observed in various cancer types or premalignant cells. However, its role in prostate cancer (PCa) development and its prognostic significance in PCa is unknown. MethodsThe mRNA and protein levels of PRKDC were analyzed in 15 pairs of PCa and benign prostatic hyperplasia tissues as well as PCa cell lines by quantitative real-time polymerase chain reaction and Western blot, respectively. Small interfering RNA and short hairpin RNA-mediated knockdown of PRKDC, followed by cell proliferation, colony formation, and soft agar assays were performed. Xenograft mouse model was used to evaluate in vivo effects of PRKDC knockdown. The association between PRKDC expression and clinicopathologic features was assessed by χ2 tests. Kaplan-Meier analysis was performed to investigate the association between PRKDC expression and overall survival. Cox proportional hazards regression models were used to examine the prognostic significance of PRKDC. ResultsExpression of PRKDC mRNA and protein was notably higher in PCa tissues and PCa cell lines. Knockdown of PRKDC markedly reduced cell proliferation, colony formation efficiency, and soft agar growth in DU145 cells. Down-regulation of PRKDC inhibited tumor growth of DU145 xenografts and enhance mice survival. In addition, PRKDC expression in PCa was significantly associated with Gleason score (P = 0.01), tumor stage (P = 0.028), and distant metastasis (P = 0.025). Patients with PCa having higher PRKDC expression had substantially shorter survival than patients with lower PRKDC expression. ConclusionDown-regulation of PRKDC attenuates tumor progression in PCa. PRKDC may potentially be a prognostic biomarker in PCa.

Dihydromyricetin from Ampelopsis grossedentata inhibits melanogenesis through down-regulation of MAPK, PKA and PKC signaling pathways

The inhibitory effects of dihydromyricetin purified from Ampelopsis grossedentata on melanogenesis and its antioxidant characteristics were investigated. Assays of tyrosinase activities and melanin content in B16F10 mouse melanoma cells were carried out spectrophotometrically, and the expression of melanogenesis-related proteins was determined by Western blotting. The possible signaling pathways involved in dihydromyricetin-mediated depigmentation were also examined using specific protein kinase regulators. The results revealed that dihydromyricetin effectively suppresses intracellular tyrosinase activity and decreases melanin amount in cells. Dihydromyricetin also exhibits antioxidant properties and effectively decreases intracellular reactive oxygen species (ROS) and reactive species (RS) levels. Our results indicated that dihydromyricetin inhibits melanogenesis through its antioxidant properties and by downregulating protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) signaling pathways. The present study indicates that dihydromyricetin has the potential to be developed into a depigmentation skin care product.

MicroRNA let-7g regulates mouse granulosa cell autophagy by targeting insulin-like growth factor 1 receptor

As an important type of somatic cell, granulosa cells play a major role in deciding the fate of follicles. Therefore, analyses of granulosa cell apoptosis and follicular atresia have become hotspots of animal research. Autophagy is a cellular catabolic mechanism that protects cells from stress conditions, including starvation, hypoxia, and accumulation of misfolded proteins. However, the relationship between autophagy and apoptosis in granulosa cells is not well known. Here, we demonstrate that let-7g regulates the mouse granulosa cell autophagy signaling pathway by inhibiting insulin-like growth factor 1 receptor expression and affecting the phosphorylation of protein kinase B/mammalian target of rapamycin. Small interference-mediated knockdown of insulin-like growth factor 1 receptor significantly promoted autophagy signaling of mouse granulosa cells. In contrast, overexpression of insulin-like growth factor 1 receptor in mouse granulosa cells attenuated autophagy activity in the presence of let-7g. In addition, overexpression of let-7g increased the apoptosis rate, as indicated by an increased number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. Finally, 3-methyladenine as well as the lysosomal enzyme inhibitor chloroquine partially blocked apoptosis. In summary, this study demonstrates that let-7g regulates autophagy in mouse granulosa cells by targeting insulin-like growth factor 1 receptor and downregulating protein kinase B/mammalian target of rapamycin signaling, and that mouse granulosa cell autophagy induced by let-7g participates in apoptosis.

MiR-24-3p Suppresses Malignant Behavior of Lacrimal Adenoid Cystic Carcinoma by Targeting PRKCH to Regulate p53/p21 Pathway.

MicroRNA (miRNA) may function as an oncogene or a tumor suppressor in tumorigenesis. However, the mechanism of miRNAs in adenoid cystic carcinoma (ACC) is unclear. Here, we provide evidence that miR-24-3p was downreglated and functions as a tumor suppressor in human lacrimal adenoid cystic carcinoma by suppressing proliferation and migration/invasion while promoting apoptosis. miR-24-3p down-regulated protein kinase C eta (PRKCH) by binding to its untranslated region (3’UTR). PRKCH increased the of the cell growth and migration/invasion in ACC cells and suppressed the expression of p53 and p21 in both mRNA and protein level. The overexpression of miR-24-3p decreased its malignant phenotype. Ectopic expression of PRKCH counteracted the suppression of malignancy induced by miR-24-3p, as well as ectopic expression of miR-24-3p rescued the suppression of PRKCH in the p53/p21 pathway. These results suggest that miR-24-3p promotes the p53/p21 pathway by down-regulating PRKCH expression in lacrimal adenoid cystic carcinoma cells.

CLK2 inhibition ameliorates autistic features associated with SHANK3 deficiency.

SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56β, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.

Down-Regulation of Protein Kinase C-ε by Prolonged Incubation with PMA Inhibits the Proliferation of Vascular Smooth Muscle Cells

Background/Aims: Phorbol myristate acetate (PMA) exerts a pleiotropic effect on the growth and differentiation of various cells. Protein kinase Cs (PKCs) plays a central role in mediating the effects of PMA on cells. The present study investigated whether the down-regulation of protein kinase C-ε (PKC-ε) is involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation caused by prolonged PMA incubation. Methods: Using cell counting, Cell Counting Kit-8 (CCK-8) and EdU incorporation assay on VSMCs, we evaluated the inhibitory effects of prolonged incubation of PMA, of lentiviruses carrying the short-hairpin RNAs (shRNA) of PKC-ε and of the PKC-ε inhibitor peptide on the proliferation and viability of cells. The effect of PKC-ε down-regulation on growth of rat breast cancer SHZ-88 cells was also measured. Results: The prolonged incubation of VSMCs with PMA for up to 72 hours resulted in attenuated cell growth rates in a time-dependent manner. The expression of PKC-ε, as assessed by Western blotting, was also decreased accordingly. Notably, the number of EdU-positive cells and the cell viability of VSMCs were decreased by shRNA of PKC-ε and the PKC-ε inhibitor peptide, respectively. The proliferation of rat breast cancer SHZ-88 cells was also attenuated by lentivirus-induced shRNA silencing of PKC-ε. Conclusions: Prolonged incubation of PMA can inhibit the expression of PKC-ε. The effect results in the inhibition of VSMC proliferation. PKC-ε silencing can also attenuate breast cancer cell growth, suggesting that PKC-ε may be a potential target for anti-cancer drugs.

Supercritical Fluid Extract of Spent Coffee Grounds Attenuates Melanogenesis through Downregulation of the PKA, PI3K/Akt, and MAPK Signaling Pathways.

The mode of action of spent coffee grounds supercritical fluid CO2 extract (SFE) in melanogenesis has never been reported. In the study, the spent coffee grounds were extracted by the supercritical fluid CO2 extraction method; the chemical constituents of the SFE were investigated by gas chromatography-mass spectrometry (GC-MS). The effects of the SFE and its major fatty acid components on melanogenesis were evaluated by mushroom tyrosinase activity assay and determination of intracellular tyrosinase activity and melanin content. The expression level of melanogenesis-related proteins was analyzed by western blotting assay. The results revealed that the SFE of spent coffee grounds (1–10 mg/mL) and its major fatty acids such as linoleic acid and oleic acid (6.25–50 μM) effectively suppressed melanogenesis in the B16F10 murine melanoma cells. Furthermore, the SFE decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). The SFE also decreased the protein expression levels of p-JNK, p-p38, p-ERK, and p-CREB. Our results revealed that the SFE of spent coffee grounds attenuated melanogenesis in B16F10 cells by downregulation of protein kinase A (PKA), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen-activated protein kinases (MAPK) signaling pathways, which may be due to linoleic acid and oleic acid.

Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3.

ResultsThe lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.ConclusionOur data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1-7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.

Arachidonic acid-induced down-regulation of protein kinase C delta in beta-cells.

We have previously identified expression of multiple protein kinase C (PKC) isoforms in insulinoma-derived beta-cells and whole islets. Both PKC delta and PKC alpha appear to be the more abundantly expressed isoforms. In this report we studied the effects of arachidonic acid (AA) on the subcellular distribution of PKC alpha and PKC delta. AA has been reported to activate both PKC alpha and PKC delta and it is thought to be an important second messenger in beta-cells. Here we report that AA interacted with and altered beta-cell pools of PKC delta preferentially over PKC alpha. AA (100 microM) over the course of 45 min reduced cytosolic levels of PKC delta (to 40 +/- 15%, compared to time zero control) leaving membrane- and cytoskeleton-associated levels near control levels. Analysis of whole cell homogenates showed a slight down-regulation of PKC delta indicating proteolysis. The down-regulation of cytosolic PKC delta appeared to be isoform specific since cytosolic PKC alpha remained at control levels over the time course. The response was dose-dependent and negligible at concentrations below 30 microM and occurred, at least partially, in the cytosolic compartment of the cell. Indomethacin also down-regulated cytosolic PKC delta preferentially over PKC alpha possibly through accumulation of AA. These findings suggest that cytosolic PKC delta may be a downstream target of this beta-cell second messenger.

Down Regulation of CLDND1 Induces Apoptosis in Breast Cancer Cells.

Identification of targets for apoptosis induction is important to provide novel therapeutic approaches in breast cancer. Our earlier studies showed that down regulation of protein kinase C δ (PKCδ) induces death in breast cancer cells. In this study we set out to identify previously unrecognized apoptosis regulators in breast cancer cells. To identify candidates, global expression analysis with microarray was performed after down regulation of PKCδ in the basal-like breast cancer cell lines MDA-MB-231, MDA-MB-468 and BT-549. Genes that were down regulated in all cell lines were further studied for survival-supporting effects. The claudin-like CLDND1 was singled out since several independent siRNAs targeting CLDND1 induced cell death in several cell lines. The cell death induced by CLDND1 knockdown was caspase-dependent, suggesting induction of apoptosis. Nuclear fragmentation, cleavage of caspase-3 and PARP and release of cytochrome C from the mitochondria upon CLDND1 depletion demonstrated involvement of the intrinsic apoptotic pathway. Inhibition of MEK1/2 and JNK further potentiated the cell death induction by CLDND1 knockdown. However, CLDND1 down regulation augmented ERK1/2 phosphorylation, which thereby may protect against the apoptosis inducing effects of CLDND1 down regulation. A concomitant inhibition of MEK1/2 suppresses the ERK1/2 phosphorylation and markedly potentiates the cell death following CLDND1 siRNA treatment. There is today little information on the function of CLDND1. These data provide novel information on CLDND1 and highlight it as a novel survival factor in basal-like breast cancer cell lines.

Inhibition of P2Y6 receptor-mediated phospholipase C activation and Ca2+ signalling by prostaglandin E2 in J774 murine macrophages

Extracellular nucleotides act as inflammatory mediators through activation of multiple purinoceptors. Under inflammatory conditions, the purinergic signalling is affected by various inflammatory mediators. We previously showed that prostaglandin (PG) E2 suppressed the elevation of intracellular Ca2+ concentration ([Ca2+]i) stimulated by P2X4, P2Y2, and P2Y6 receptors in J774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y6 receptor-mediated function in J774 cells. The P2Y6 receptor agonist UDP induced a sustained elevation of [Ca2+]i by stimulating the phospholipase C (PLC) signalling pathway. PGE2 inhibited [Ca2+]i elevation and phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. J774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gs-coupled receptor. PGE2 and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in J774 cells. The inhibitory effects of PGE2 on P2Y6 receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE2-induced inhibition of Ca2+ response and PI hydrolysis could not be mimicked by a lipophilic cAMP derivative, dibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE2 was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE2 inhibited PLC activation induced by aluminium fluoride, but not by the Ca2+-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE2 was impaired by Gs knockdown using siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the Gq/11-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism.

Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs) although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB), we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ) expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs as well as late osteogenic markers osteopontin (OPN), osteocalcin (OCN) and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

Potassium Channels and Uterine Vascular Adaptation to Pregnancy and Chronic Hypoxia

During a normal course of pregnancy, uterine vascular tone is significantly decreased resulting in a striking increase in uterine blood flow, which is essential for fetal development and fetal growth. Chronic hypoxia during gestation may adversely affect the normal adaptation of uterine vascular tone and increase the risk of preeclampsia and fetal intrauterine growth restriction. In this review, we present evidence that the regulation of K+ channels is an important mechanism in the adaptation of uterine vascular tone to pregnancy and hypoxia. There are four types of K+ channels identified in arterial smooth muscle cells: 1) voltage-dependent K(+) (Kv) channels, 2) Ca2+-activated K(+) (KCa) channels, 3) inward rectifier K(+) (KIR) channels, and 4) ATP-sensitive K(+) (KATP) channels. Pregnancy differentially augments the expression and activity of K(+) channels via downregulation of protein kinase C signaling in uterine and other vascular beds, leading to decreased uterine vascular tone and increased uterine blood flow. Sex steroid hormones play an important role in the pregnancy- mediated alteration of K+ channels in the uterine vasculature. In addition, chronic hypoxia alters uterine vascular K(+) channels expression and activities via modulation of steroid hormones/receptors-mediated signaling, resulting in increased uterine vascular tone during pregnancy.

Short- and Long-Term Regulation of Intestinal Na+/H+Exchange Activity Associated with TLR2 Receptor Activation Is Independent of Nuclear Factor-κB Signaling

Type 2 Toll-like receptors (TLR2s) are expressed in cell membranes and recognize a wide range of pathogen-associated molecular patterns derived from bacteria, such as lipoteichoic acid (LTA). The aim of this study was to evaluate the effect of TLR2 activation by LTA on the activity of type 1 Na(+)/H(+) exchanger (NHE) in T84 intestinal epithelial cells. Short-term (0.5 hour) and long-term (18 hours) TLR2 activation significantly inhibited NHE1 activity in a concentration-dependent manner (0.01-100 µg/ml; -7 ± 3 to -21 ± 3% and 3 ± 3 to -21 ± 3% of control values, respectively). S3226 [3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride], an NHE3-selective inhibitor, did not affect the inhibitory effect on NHE activity. LTA-induced NHE inhibition did not occur in the presence ofethylisopropylamiloride (an NHE1 inhibitor). Long-term TLR2 activation decreased NHE1 affinity for Na(+) (Km= 64.98 ± 1.67 mM) compared with control (Km= 20.44 ± 0.54 mM) without changes in Vmax values. After TLR2 activation, we observed tyrosine-protein kinase (SRC) activation, phosphatidylinositol 3-kinase (PI3K) recruitment, and adenylyl cyclase (AC3) phosphorylation. The total amount of AC3 increased (23 ± 8% of control) after long-term treatment with LTA. Anti-AC3 small interfering RNA prevented LTA-induced NHE1 inhibition, similar to that observed with the AC3 inhibitor KH7 [(±)-2-(1H-benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide]. A significant increase in cAMP levels (32 ± 3% and 14 ± 2% after short- and long-term stimulation, respectively) was detected, and inhibition of protein kinase A (PKA), phospholipase C (PLC), and downregulation of protein kinase C (PKC) prevented NHE1 inhibition. Inhibition of nuclear factor-κΒ (NF-κB) failed to revert NHE1 inhibition. We concluded that activation of TLR2 reduces NHE1 activity in epithelial cells through an alternative pathway that is unrelated to NF-κB, which involves SCR, PI3K, AC3, PKA, PLC, and PKC.

Elevated dopamine D2 receptor in prefrontal cortex of CUMS rats is associated with downregulated cAMP-independent signaling pathway

Because depression is associated with significant morbidity and functional disability, it is important to reveal the mechanism of action. A variety of studies have suggested the involvement of dopaminergic receptors in the pathophysiological mechanism of non-stress-associated depression-like behavior in rodents. Nevertheless, controversy exists about whether chronic stress acts on dopaminergic receptors in the prefrontal cortex. Thus, we investigated the level of dopamine D2 receptors (DRD2) and the possible mechanisms involved in a chronic unpredictable mild stress (CUMS) rat model of depression. The results showed CUMS-induced, depression-like symptoms in the rat, characterized by reduced sucrose consumption and body mass, and increased duration of immobility in a forced swimming test. Moreover, chronic stress upregulated the expression of DRD2 but downregulated protein kinase A (PKA), transcription factor cAMP response element binding protein (CREB), and phospho-CREB (p-CREB) in the prefrontal cortex, as demonstrated by Western blot. Notably, in the rat model of depression, decreased cyclic adenine monophosphate (cAMP) levels and PKA activity were present at the same time, which is consistent with clinical findings in depressed patients. Our findings suggested that dopaminergic system dysfunction could play a central role in stress-related disorders such as depression.

Tumor-induced STAT3 hyperactivation creates a dendritic cell differentiation block by significantly downregulating protein kinase C βII expression (P2003)

Abstract Tumor mediated blockade of dendritic cell (DC) differentiation is a large component of cancer induced immunosuppression, contributing to tumor outgrowth. Tumors impair DC differentiation via factors that hyperactivate STAT3 in DC progenitors, though the mechanisms by which this occurs are largely unknown. PKCβII is essential in DC differentiation, and we test here if tumor driven STAT3 hyperactivation downregulates PKCβII expression, and if this is the mechanism underlying impaired DC differentiation. Myeloid cells from tumor bearing mice have significantly decreased PKCβII expression. Culture in tumor conditioned media (TCM) significantly decreased PKCβII expression and significantly impaired phorbol ester driven DC differentiation in human monocytes and in a human cell line model (KG1). In KG1, this was dependent on PKCβII downregulation: enforced PKCβII expression preserved phorbol ester driven DC differentiation in the face of TCM. Interestingly, PKCβII overexpression and/or activation also antagonized TCM-driven STAT3 activation. STAT3 drives this decreased PKCβII expression: TCM induced STAT3 activation and drove 4.5 fold higher STAT3 binding to the PKCβ promoter (compared to media control, p&amp;lt;0.001). Mutation of a STAT3 consensus binding site in the promoter eliminates the ability of STAT3 to impair PKCβ promoter activity. Together, these observations argue that tumor driven STAT3 hyperactivation inhibits DC differentiation by downregulating PKCβII.

Over-expression of calpastatin aggravates cardiotoxicity induced by doxorubicin

Doxorubicin causes damage to the heart, which may present as cardiomyopathy. However, the mechanisms by which doxorubicin induces cardiotoxicity remain not fully understood and no effective prevention for doxorubicin cardiomyopathy is available. Calpains, a family of calcium-dependent thiol-proteases, have been implicated in cardiovascular diseases. Their activities are tightly controlled by calpastatin. This study employed transgenic mice over-expressing calpastatin to investigate the role of calpain in doxorubicin-induced cardiotoxicity. Doxorubicin treatment decreased calpain activities in cultured neonatal mouse cardiomyocytes and in vivo mouse hearts, which correlated with down-regulation of calpain-1 and calpain-2 proteins. Over-expression of calpastatin or incubation with pharmacological calpain inhibitors enhanced apoptosis in neonatal and adult cardiomyocytes induced by doxorubicin. In contrast, over-expression of calpain-2 but not calpain-1 attenuated doxorubicin-induced apoptosis in cardiomyocytes. The pro-apoptotic effects of calpain inhibition were associated with down-regulation of protein kinase B (AKT) protein and mRNA expression, and a concomitant reduction in glycogen synthase kinase-3beta (GSK-3β) phosphorylation (Ser9) in doxorubicin-treated cardiomyocytes. Blocking AKT further increased doxorubicin-induced cardiac injuries, suggesting the effects of calpain inhibition may be mediated by inactivating the AKT signalling. In an in vivo model of doxorubicin-induced cardiotoxicity, over-expression of calpastatin exacerbated myocardial dysfunction as assessed by echocardiography and haemodynamic measurement in transgenic mice 5 days after doxorubicin injection. The 5-day mortality was higher in transgenic mice (29.16%) compared with their wild-type littermates (8%) after doxorubicin treatment. Over-expression of calpastatin enhances doxorubicin-induced cardiac injuries through calpain inhibition and thus, calpains may protect cardiomyocytes against doxorubicin-induced cardiotoxicity.