Fibronectin increases matrix metalloproteinase 9 expression through activation of c-Fos via extracellular-regulated kinase and phosphatidylinositol 3-kinase pathways in human lung carcinoma cells

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.     

Induction of cyclooxygenase-2 synthesis by ligation of the macrophage alpha(2)-macroglobulin signalling receptor

We have studied the induction of cyclooxygenase-2 (COX-2) in macrophages consequent to ligating the alpha(2)-macroglobulin (alpha(2)M) signalling receptor (alpha(2)MSR) with receptor-recognized forms of alpha(2)M (alpha(2)M*). Macrophage stimulation with alpha(2)M* increased total cellular and nuclear COX-2 two- to threefold. The maximal increase in COX-2 occurred at a ligand concentration of 50-100 pM and after 2 h. Modulation of intracellular Ca(2+) levels or incubation of [35S] methionine-labelled macrophages with actinomycin D, prior to treatment with alpha(2)M*, markedly reduced the induction of total cellular and nuclear COX-2. Protein kinase C (PKC) or phospholipase A(2) (PLA(2)) inhibition in alpha(2)M*-stimulated macrophages or inhibition of the p21(ras)-dependent mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI 3-kinase) signalling pathways also significantly reduced alpha(2)M*-induced total cellular and nuclear COX-2 expression. Thus, COX-2 induction is dependent on cPLA(2) activity, Ca(2+) mobilization, and PKC activity and requires participation of both the p21(ras)-dependent MAPK and PI 3-kinase signalling pathways. COX-2 activation may mediate alpha(2)M*-induced mitogenesis, which we have previously observed in this and other cell types.     

Activation of signaling pathways and regulatory mechanisms of mRNA translation following myocardial ischemia-reperfusion.

Protein expression in the heart is altered following periods of myocardial ischemia. The changes in protein expression are associated with increased cell size that can be maladaptive. There is little information regarding the regulation of protein expression through the process of mRNA translation during ischemia and reperfusion in the heart. Therefore, the purpose of this study was to identify changes in signaling pathways and downstream regulatory mechanisms of mRNA translation in an in vivo model of myocardial ischemia and reperfusion. Hearts were collected from rats whose left main coronary arteries had either been occluded for 25 min or reversibly occluded for 25 min and subsequently reperfused for 15 min. Following reperfusion, both the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways were activated, as evidenced by increased phosphorylation of Akt (PKB), extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase. Activation of Akt stimulated signaling through the protein kinase mammalian target of rapamycin, as evidenced by increased phosphorylation of two of its effectors, the ribosomal protein S6 kinase and the eukaryotic initiation factor eIF4E binding protein 1. Ischemia and reperfusion also resulted in increased phosphorylation of eIF2 and eIF2B. These changes in protein phosphorylation suggest that control of mRNA translation following ischemia and reperfusion is modulated through a number of signaling pathways and regulatory mechanisms.     

Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion

Tumor cyclooxygenase-2 (COX-2) expression is known to be associated with enhanced tumor invasiveness. In the present study, we evaluated the importance of the COX-2 product prostaglandin E2 (PGE2) and its signaling through the EP4 receptor in mediating non-small cell lung cancer (NSCLC) invasiveness. Genetic inhibition of tumor COX-2 led to diminished matrix metalloproteinase (MMP)-2, CD44, and EP4 receptor expression and invasion. Treatment of NSCLC cells with exogenous 16,16-dimethylprostaglandin E2 significantly increased EP4 receptor, CD44, and MMP-2 expression and matrigel invasion. In contrast, anti-PGE2 decreased EP4 receptor, CD44, and MMP-2 expression in NSCLC cells. EP4 receptor signaling was found to be central to this process, because antisense oligonucleotide-mediated inhibition of tumor cell EP4 receptors significantly decreased CD44 expression. In addition, agents that increased intracellular cAMP, as is typical of EP4 receptor signaling, markedly increased CD44 expression. Moreover, MMP-2-AS treatment decreased PGE2-mediated CD44 expression, and CD44-AS treatment decreased MMP-2 expression. Thus, PGE2-mediated effects through EP4 required the parallel induction of both CD44 and MMP-2 expression because genetic inhibition of either MMP-2 or CD44 expression effectively blocked PGE2-mediated invasion in NSCLC. These findings indicate that PGE2 regulates COX-2-dependent, CD44- and MMP-2-mediated invasion in NSCLC in an autocrine/paracrine manner via EP receptor signaling. Thus, blocking PGE2 production or activity by genetic or pharmacological interventions may prove to be beneficial in chemoprevention or treatment of NSCLC.     

Prostaglandin E2 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases

Prostaglandins (PGs) have been implicated in lowering intraocular pressure (IOP). A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing impaired COX-2 expression in the non-pigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. The present study investigates the effect of the major COX-2 product, PGE(2), on the expression of its synthesizing enzyme in human NPE cells (ODM-2). PGE(2) led to an increase of COX-2 mRNA and protein expression, whereas the expression of COX-1 remained unchanged. Upregulation of COX-2 expression by PGE(2) was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, and was abrogated by inhibitors of both pathways. Moreover, PGE(2)-induced COX-2 expression was suppressed by the intracellular calcium chelator, BAPTA/AM, and the protein kinase C inhibitor bisindolylmaleimide II, whereas the protein kinase A inhibitor H-89 was inactive in this respect. Induction of COX-2 expression was also elicited by butaprost (EP(2) receptor agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) receptor agonist), but not by EP(1)/EP(3) receptor agonists (17-phenyl-omega-trinor PGE(2), sulprostone). Consistent with these findings, the EP(1)/EP(2) receptor antagonist, AH-6809, and the selective EP(4) receptor antagonist, ONO-AE3-208, significantly reduced PGE(2)-induced COX-2 expression. Collectively, our results demonstrate that PGE(2) at physiologically relevant concentrations induces COX-2 expression in human NPE cells via activation of EP(2)- and EP(4) receptors and phosphorylation of p38 and p42/44 MAPKs. Positive feedback regulation of COX-2 may contribute to the production of outflow-facilitating PGs and consequently to regulation of IOP.     

Adenosine triphosphate regulates NADPH oxidase activity leading to hydrogen peroxide production and COX-2/PGE2 expression in A549 cells

Non-small cell lung carcinoma (NSCLC) accounts for most of all lung cancers, which is the leading cause of mortality in human beings. High level of cyclooxygenase-2 (COX-2) is one of the features of NSCLC and related to the low survival rate of NSCLC. However, whether extracellular nucleotides releasing from stressed resident tissues contributes to the expression of COX-2 remains unclear. Here, we showed that stimulation of A549 cells by adenosine 5'-O-(3-thiotriphosphate) (ATPγS) led to an increase in COX-2 gene expression and prostaglandin E(2) (PGE(2)) synthesis, revealed by Western blotting, RT-PCR, promoter assay, and enzyme-linked immunosorbent assay. In addition, ATPγS induced intracellular reactive oxygen species (ROS) generation through the activation of NADPH oxidase. The increase of ROS level resulted in activation of the c-Src/epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor (NF)-κB cascade. We also found that activated Akt was translocated into the nucleus and recruited with NF-κB and p300 to form a complex. Thus, activation of p300 modulated the acetylation of histone H4 via the NADPH oxidase/c-Src/EGFR/PI3K/Akt/NF-κB cascade stimulated by ATPγS. Our results are the first to show a novel role of NADPH oxidase-dependent Akt/p65/p300 complex formation that plays a key role in regulating COX-2/PGE(2) expression in ATPγS-treated A549 cells. Taken together, we demonstrated that ATPγS stimulated activation of NADPH oxidase, resulting in generation of ROS, which then activated the downstream c-Src/EGFR/PI3K/Akt/NF-κB/p300 cascade to regulate the expression of COX-2 and synthesis of PGE(2) in A549 cells. Understanding the regulation of COX-2 expression and PGE(2) release by ATPγS on A549 cells may provide potential therapeutic targets of NSCLC.     

p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.     

Regulation of vascular endothelial cell growth factor expression in mouse mammary tumor cells by the EP2 subtype of the prostaglandin E2 receptor

Prostaglandin E(2) (PGE(2)), a major metabolite of the cyclooxygenase pathway in the mammary gland, induces angiogenesis during mammary tumor progression. To better define the molecular mechanisms involved, we examined the role of the G protein-coupled receptors (GPCR) for PGE(2) in mammary tumor cell lines isolated from MMTV-cyclooxygenase-2 (COX-2) transgenic mice. Expression of the EP2 subtype of the PGE(2) receptor was correlated with the tumorigenic phenotype and the ability to induce vascular endothelial growth factor (VEGF). Overexpression of EP2 by adenoviral transduction into EP2-null cells resulted in the induction of VEGF expression in response to PGE(2) and CAY10399, an EP2 receptor agonist. The induction of VEGF by the EP2 receptor did not require the hypoxia inducible factor (HIF)-1alpha pathway, MAP kinase pathway, or phosphoinositide-3-kinase/Akt pathway, but required the cAMP/protein kinase A pathway. These results suggest that EP2 receptor is a critical element for PGE(2) mediated VEGF induction in mouse mammary tumor cells.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

PKC-delta/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.     

Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2alpha

Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (-70/-29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2alpha (AP-2alpha), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2alpha synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (-103/-29) with Sp1 and AP-2alpha expression plasmids showed that Sp1 and AP-2alpha synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2alpha alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (-70/-29), whereas AP-2alpha bound to a more upstream promoter region (-103/-71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2alpha may be mediated by the binding of Sp1 to the promoter region (-70/-29) and the interaction with AP-2alpha, which binds to the promoter region (-103/-71).