Quercetin inhibits transcriptional up-regulation of histamine H1 receptor via suppressing protein kinase C-δ/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 signaling pathway in HeLa cells

It has been reported that the histamine H₁ receptor (H1R) gene is up-regulated in patients with allergic rhinitis and H1R expression level strongly correlates with the severity of allergy symptoms. Accordingly compounds that suppress the H1R gene expression are promising as useful anti-allergic medications. Recently, we demonstrated that histamine or phorbol-12-myristate-13-acetate (PMA) stimulation induced the up-regulation of H1R gene expression through the protein kinase Cδ (PKCδ)/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 signaling pathway in HeLa cells expressing H1R endogenously. Quercetin is one of the well-characterized flavonoids and it possesses many biological activities including anti-allergic activity. However, effect of quercetin on H1R signaling is remained unknown. In the present study, we examined the effect of quercetin on histamine- and PMA-induced up-regulation of H1R gene expression in HeLa cells. We also investigated its in vivo effects on the toluene-2,4-diisocyanate (TDI)-sensitized allergy model rats. Quercetin suppressed histamine- and PMA-induced up-regulation of H1R gene expression. Quercetin also inhibited histamine- or PMA-induced phosphorylation of Tyr³¹¹ of PKCδ and translocation of PKCδ to the Golgi. Pre-treatment with quercetin for 3 weeks suppressed TDI-induced nasal allergy-like symptoms and elevation of H1R mRNA in the nasal mucosa of TDI-sensitized rats. These data suggest that quercetin suppresses H1R gene expression by the suppression of PKCδ activation through the inhibition of its translocation to the Golgi.     

Pharmacology of α7 nicotinic acetylcholine receptor mediated extracellular signal-regulated kinase signalling in PC12 cells

Background and purpose:

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate cell survival and memory processing. The involvement of specific nAChR subtypes in downstream signalling events has been ill defined thus far, because of a lack of subtype-selective ligands. In this study, we investigated activation and modulation of α7 nAChR-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) in PC12 cells, using selective agonists and positive allosteric modulators.

Experimental approach:

We used undifferentiated PC12 cells endogenously expressing α7 nAChR for both biochemical and functional studies. ERK phosphorylation changes were measured by using a novel In-Cell Western procedure. α7 nAChR-mediated Ca²⁺ signalling was determined by using the fluorometric imaging plate reader assay.

Key results:

Robust induction of ERK phosphorylation followed exposure of PC12 cells to the selective agonist PNU-282987 in the presence of the α7 nAChR modulator PNU-120596. ERK phosphorylation was transient and was attenuated by the selective antagonist methyllycaconitine. Consistent with allosteric modulation of α7 nAChRs, PNU-120596 enhanced both the agonist potency and efficacy in activating ERK. Moreover, α7 nAChR agonists could be quantitatively differentiated based on their potency in activating ERK signalling. The rank order of potencies correlated fairly well with the corresponding binding K_i values of these α7 nAChR agonists.

Conclusions and implications:

The present work extends previous observations demonstrating the involvement of α7 nAChRs in ERK1/2 phosphorylation in PC12 cells. The In-Cell Western procedure allowed a detailed investigation of α7 nAChR function and downstream ERK signalling in response to agonist and allosteric modulators.     

Effect of inhibition of extracellular signal-regulated kinase on relaxations to β-adrenoceptor agonists in porcine isolated blood vessels

Background and purpose:

Stimulation of vascular β-adrenoceptors causes vasodilatation through activation of adenylyl cyclase (AC) and plasma membrane potassium channels, and β-adrenoceptors have been linked to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in various cell lines. However, how these findings relate to functional responses in intact tissues is largely unknown. The aim of this study, therefore, was to investigate the role of ERK in β-adrenoceptor-induced vasodilatation.

Experimental approach:

Segments of porcine coronary artery were mounted in a Mulvany wire myograph and bathed in Krebs–Henseleit buffer gassed with 95% O₂/5% CO₂ and maintained at 37°C. Tissues were pre-contracted with the thromboxane mimetic U46619, endothelin-1 or KCl. Cumulative concentration–response curves to β-adrenoceptor agonists or forskolin were then carried out in the absence or presence of the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 (10 or 50 µM) or U0126 (10 µM).

Key results:

PD98059 caused a concentration-dependent leftward shift in response to isoprenaline (pEC₅₀ control, 7.5 ± 0.1; 50 µM PD98059, 8.1 ± 0.1: P < 0.05). Inhibition of MEK also enhanced the maximum relaxation seen with salbutamol, but not the responses to the β₁-adrenoceptor selective agonist xamoterol or the AC activator forskolin. There was no enhancement of the relaxations to β-adrenoceptor agonists after inhibition of ERK activation in tissues pre-contracted with KCl or treated with the K⁺ channel blocker tetraethylammonium.

Conclusions and implications:

These data indicate that ERK inhibits β₂-adrenoceptor-mediated vasodilatation through a mechanism which may involve inactivation of plasma membrane potassium channels.     

Rho-kinase inhibitors decrease TGF-β-stimulated VEGF synthesis through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We have previously reported that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In order to investigate whether Rho-kinase is involved in the TGF-β-stimulated VEGF synthesis in these cells we examined the effects of Rho-kinase inhibitors on the VEGF synthesis. TGF-β time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1) which is a well known substrate of Rho-kinase. Y27632 and fasudil, Rho-kinase inhibitors, significantly reduced the TGF-β-stimulated VEGF synthesis as well as the MYPT-1 phosphorylation. Y27632 and fasudil failed to affect the TGF-β-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or Smad2. On the contrary, Y27632 as well as fasudil markedly suppressed the TGF-β-induced phosphorylation of SAPK/JNK. Taken together, our results strongly suggest that Rho-kinase regulates TGF-β-stimulated VEGF synthesis via SAPK/JNK activation in osteoblasts.     

Retinoic acid remodels extracellular matrix (ECM) of cultured human fetal palate mesenchymal cells (hFPMCs) through down-regulation of TGF-β/Smad signaling

The regulation of extracellular matrix (ECM) by retinoic acid (RA) is interesting in light of the fact that the ECM plays an essential role in morphogenesis and palatal shelf elevation. In the current study, we explored the effect of RA overexposure on ECM and the probable mechanisms in cultured human fetal palate mesenchymal cells (hFPMCs). RA dose-dependently inhibited cell proliferation and mRNA and protein levels of ECM components fibronectin, tenascin C and fibrillin-2. Zymography revealed that MMP-2 activity was suppressed by RA. Further analysis revealed that mRNA levels of MMP2 and TIMP2 were decreased, while the MMP2/TIMP2 mRNA ratio was increased, which might facilitate the ECM degradation. Because of the pivotal role of TGF-β/Smad pathway in palatogenesis we therefore checked the effect of RA on TGF-β/Smad signaling. The results indicated RA treatment increased Smad7 expression and decreased the levels of TGF-β1, TGF-β3, TGF-β type II receptor (TβRII) and phosphorylated Smad2 and Smad3. Activation of the Smad pathways by either exogenous TGF-β3 or recombinant adenoviruses for Smad3 attenuated RA-induced inhibition of cell proliferation and ECM components and rescued the RA-altered MMP2/TIMP2 mRNA ratio. In conclusion, these findings suggested that RA overexposure inhibited cell proliferation and disrupted the ECM network through down-regulation of TGF-β/Smad pathway.     

Role of extracellular signal—regulated kinase in free radical—induced injury in kidney of rats treated with cephaloridine

We examined the role of a down stream of intracellular signaling pathway,extracellular signal-regulated kinase(ERK),in cephaloridine (CER)-induced nephrotoxicity in rats.The increase in phosphorylated ERK(pERK,activated ERK) was detected in nucleus fraction prepared from rat kidney cortex 24h after injections of antibiotic CER with the increase in BUN level.The slices prepared from rat kidney cortex were incubated in the medium containing PD980-59,a MEK1/2 inhibitor,for the measurement of free radical production and cell injure(LDH leakage).CER caused not only the increases in lipid peroxidation as an index of free radical production and LDH leakage,but also ERK activation in nucleus fraction.MEK1/2 inhibitor ameliorated CER-induced injury and suppressed ERK activation in the slices.These results suggest a possible role of MEK/ERK signaling pathway in free radical-induced CER nephrotoxicity.     

A physiological concentration of glucocorticoid inhibits the pro-inflammatory cytokine-induced proliferation of adult rat cardiac fibroblasts: roles of extracellular signal-regulated kinase 1/2 and nuclear factor-κB

1. Inflammation‐induced proliferation of cardiac fibroblasts plays an important role in cardiac remodelling. Pharmacological doses of exogenous glucocorticoids (GC) are the most effective therapy for inflammatory diseases. Similarly, physiological concentrations of endogenous GC have recently been shown to have anti‐inflammatory effects. Therefore, the aim of the present study was to determine whether a physiological concentration of GC could inhibit pro‐inflammatory cytokine‐stimulated proliferation of cardiac fibroblasts and to explore the mechanisms involved. 2. Cardiac fibroblasts were isolated from adult male Sprague‐Dawley rats and cell proliferation was measured using a CCK‐8 kit. Western blotting was used to detect protein expression of extracellular‐regulated kinase (ERK) 1/2 and nuclear factor (NF)‐κB. 3. Cardiac fibroblast proliferation was significantly increased by tumour necrosis factor‐α, interleukin (IL)‐1β and angiotensin II and was accompanied by upregulated protein expression of ERK1/2 and NF‐κB. A physiological concentration of hydrocortisone (127 ng/mL) not only inhibited the proliferation of cardiac fibroblasts, but also suppressed activation of ERK1/2 and NF‐κB. These effects of hydrocortisone were abrogated by the glucocorticoid receptor (GR) antagonist RU‐486 (100 nmol/L). Furthermore, inflammation‐induced cardiac fibroblast proliferation was also blocked by the mitogen‐activated protein kinase kinase 1/2 inhibitor U0126 (100 nmol/L) and the NF‐κB inhibitor pyrrolidine dithiocarbamate (1 μmol/L). Cytokine‐induced ERK1/2 phosphorylation and cyclin D1 expression were attenuated by U0126, suggesting that the ERK1/2 and NF‐κB signalling pathways were involved in cardiac fibroblast proliferation. 4. In conclusion, the results of the present study indicate that a physiological concentration of hydrocortisone can inhibit inflammation‐induced proliferation of cardiac fibroblasts by preventing the activation of ERK1/2 and NF‐κB.     

Endothelin—1 promoted proliferation of vascular smooth muscle cell through pathway of extracellular signal—regulated kinase and cyclin D1

目的:探讨内皮素-1是否通过细胞周期蛋白质D1与细胞外调节蛋白激酶通路促进人脐动脉平滑肌细胞增殖。方法:采用MTT法观察ET-1和PD98059对人脐动脉平滑肌细胞生长的作用;[~3H]TdR法观察对细胞DNA合成的作用;流式细胞仪法观察对细胞增殖周期的影响;蛋白质印迹法观察对细胞外调节蛋白激酶和细胞周期蛋白质D1表达的影响。结果:首先,同没有ET-1组和PD98059组比较,ET-1促进平滑肌细胞增殖(P<0.05)。PD98059抑制ET-1诱导的血管平滑肌细胞增殖。第二,与没有ET-1组比较,ET-1促进平滑肌细胞DNA合成(P<0.05)。第三,ET-1促进平滑肌细胞增殖周期从G_0/G_1期向S期的转变,与没有ET-1组比较,G_0/G_1期细胞百分比明显减少,S期细胞百分比明显增加(P<0.05)。第四,ET-1增加细胞外信号调节性激酶的磷酸化水平和细胞周期蛋白质D1的蛋白表达,ERK的抑制剂可以抑制细胞外信号调节性激酶的磷酸化水平和细胞周期蛋白质D1的蛋白表达,与没有ET-1组比较,磷酸化-ERK和细胞周期蛋白质D1表达明显增强,对非磷酸化ERK表达没有影响。结论:内皮素-1可以通过细胞周期调节素D1与细胞外信号调节性激酶通路促进平滑肌细胞增殖。     

Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes

By secreting proteases such as cathepsins and matrix metalloproteinases (MMPs), macrophage foam cells may be a major cause of ruptured atherosclerotic plaques. The aims of the present study were to investigate in vitro role of human macrophage foam cells in degrading type I collagen, a major component of extracellular matrix (ECM) in plaques, and to establish whether the pro-inflammatory molecules, tumor necrosis factor (TNF)-alpha, and receptor activator of nuclear factor-κB ligand (RANK-L) increase this degradation. CD14+ monocytes isolated from peripheral blood were differentiated into macrophage foam cells and cultured on a type I collagen matrix in the presence of TNF-alpha and RANK-L. Matrix degradation was measured by the cathepsin K-generated C-terminal cross-linked telopeptide of type I collagen (CTX-I) and the MMP-generated carboxyterminal telopeptide of type I collagen (ICTP) in supernatants showing that macrophage foam cells secrete MMPs and cathepsin K, resulting in release of ICTP and CTX-I. Stimulation with TNF-alpha increased CTX-I and ICTP dose dependently, with ICTP levels increasing by 59% and CTX-I levels increasing by 43%. RANK-L enhanced the release of CTX-I and ICTP by 56% and 72%, respectively. This is, to our knowledge, the first data describing a simple in vitro system in which macrophage foam cells degradation of matrix proteins can be monitored. This degradation can be enhanced by cytokines since TNF-alpha and RANK-L significantly increased the matrix degradation. This in vitro system in part is a model system for the macrophage-mediated proteolytic degradation of the ECM, which is found in many diseases with an inflammatory component.     

Gastrointestinal gene delivery by cyclodextrins – In vitro quantification of extracellular barriers

Local gene delivery represents a promising therapeutic approach for diseases of the intestine. However, the gastrointestinal tract poses significant challenges to successful gene delivery. Cyclodextrins (CDs) have been extensively investigated as non-viral vectors. Here, we assessed the suitability of an amphiphilic cationic CD for intestinal gene transfer, with particular focus on extracellular barriers.     

Upregulation of Sirt1 by tyrosol suppresses apoptosis and inflammation and modulates extracellular matrix remodeling in interleukin-1β-stimulated human nucleus pulposus cells through activation of PI3K/Akt pathway

Intervertebral disc degeneration (IDD) is the major pathogenesis of lower back pain. Tyrosol is a polyphenolic compound that exhibits anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Herein, we explored the effects and mechanisms of tyrosol on IDD progression in interleukin (IL)-1β-stimulated human nucleus pulposus cells (HNPCs). Cell viability and apoptosis were detected by CCK-8 and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was examined to evaluate inflammation. The mRNA expression of matrix metalloproteinases (MMPs) (MMP-3/9/13), collagen type II, SRY-related high mobility group box 9 (SOX-9), and aggrecan was measured by qRT-PCR. Protein levels of silent information regulator 2 homolog 1 (Sirt1), phosphorylated protein kinase B (p-Akt), Akt, collagen type II, SOX-9, and aggrecan were determined by western blot. Results showed that tyrosol attenuated IL-1β-induced viability reduction, apoptosis, and caspase-3/7 activity in HNPCs. The increase in the production of TNF-α, IL-6, NO, and PGE2 in IL-1β-treated HNPCs was abolished by tyrosol treatment. Tyrosol treatment reversed IL-1β-induced upregulation of MMP-3, MMP-9, and MMP-13, and downregulation of collagen II, SOX-9, and aggrecan in HNPCs. Additionally, tyrosol treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in IL-1β-stimulated HNPCs. Sirt1 was upregulated by tyrosol, and Sirt1 silencing inhibited Akt phosphorylation in HNPCs. Sirt1 knockdown attenuated the effects of tyrosol on IL-1β-induced apoptosis, inflammation, and ECM remodeling in HNPCs. In summary, upregulation of Sirt1 by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1β-stimulated HNPCs through activation of PI3K/Akt pathway.     

cAMP: fuel for extracellular adenosine formation?

It is well known that cAMP, an important intracellular second messenger, is released from many cells upon adenylate cyclase stimulation. Cell surface bound phosphodiesterase together with ecto-5'-nucleotidase may convert the extracellular cAMP to adenosine, which may stimulate in a paracrine and/or autocrine manner cells expressing P1 receptors. In this issue of the British Journal of Pharmacology, Chiavegatti et al. demonstrate the existence of an extracellular cAMP-adenosine cascade in skeletal muscle cells which suggests a link between adrenergic stimulation of contraction, elevated cAMP formation and release and exercise hyperaemia.     

Protein kinase Cδ mediates the activation of protein kinase D2 in platelets

Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCv in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a G_q inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is G_q-, but not G_12/13-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ- or PKC?-deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and G_q-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in platelets.     

17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway

We hypothesized that 17β-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway. Rat aortic rings were contracted with cumulative addition of U46619, NaF, KCl or PDBu 30 min after pretreatment with 17β-estradiol (10, 30, and 100 μM) or vehicle. We measured the amount of GTP RhoA and the level of phosphorylation of the myosin light chain (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Pretreatment with 17β-estradiol dose-dependently inhibited the concentration-response curves in response to U46619, NaF or KCl, but not to PDBu. 17β-Estradiol decreased not only the level of phosphorylation of MYPT1^Thr855 and CPI17^Thr38 as well as MLC₂₀, but also the activity of RhoA induced by U46619 or NaF. However, 17β-estradiol did not affect the level of phosphorylation of CPI17 induced by PDBu. 17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.