Miguel A. Iñiguez

An Essential Role of the Nuclear Factor of Activated T Cells in the Regulation of the Expression of the Cyclooxygenase-2 Gene in Human T Lymphocytes

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions −117 and −58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca2+/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

Cyclooxygenase-2: a therapeutic target in angiogenesis

Angiogenesis has a role in the pathogenesis of several disorders, including cancer, chronic inflammatory diseases and retinopathies. Recent evidence demonstrates that the production of prostanoids by cyclooxygenase-2 (COX-2) promotes the expression of pro-angiogenic factors. Furthermore, inhibition of COX-2 by non-steroidal anti-inflammatory drugs leads to restricted angiogenesis and downregulated production of pro-angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor. These findings suggest that COX enzymes could be important therapeutic targets in the treatment of pathological angiogenesis.

The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression

Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell-dependent (NF-AT-dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.

Neonatal hypothyroidism affects the timely expression of myelin-associated glycoprotein in the rat brain.

Congenital hypothyroidism strongly affects myelination. To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the steady state levels of myelin-associated glycoprotein (MAG) and its mRNA in rat brain during the first postnatal month. As studied by immunoblot analysis of several brain regions, MAG increased from days 10-15 onwards, reaching constant levels by days 20-25. Hypothyroid samples showed a delay in the accumulation of MAG that was more severe in rostral regions, such as cortex and hippocampus. The effect of hypothyroidism on the accumulation of the protein correlated with mRNA levels. MAG mRNA started to accumulate in the cerebrum of normal animals by postnatal day 7, reaching maximal levels by day 20. Hypothyroid rats showed a delay of several days in the onset of mRNA expression, increasing thereafter at the same rate as in normal animals, and eventually reaching similar values. When individual brain regions were analyzed, we found strong regional differences in the effect of hypothyroidism. The cerebral cortex was most affected, with messenger levels lower than in normal animals at all ages. In more caudal regions differences between control and hypothyroid rats were evident only at the earlier stages of myelination, with spontaneous recovery at later ages. By run on analysis, we found no differences in transcriptional activities of the MAG gene in normal, hypothyroid, or T4-treated rats. Therefore, the effects of hypothyroidism on MAG mRNA and protein levels were most likely caused by decreased mRNA stability. We propose that thyroid hormone contributes to enhanced myelin gene expression by affecting the stability of newly transcribed mRNA in the early phases of myelination.

Human Vascular Smooth Muscle Cells but Not Endothelial Cells Express Prostaglandin E Synthase

In a previous work, we postulated that endothelial cells possess only the following 2 enzymes involved in prostanoid synthesis: cyclooxygenase and prostacyclin synthase. The present work focused on investigating the expression of prostaglandin (PG) E synthase (PGES) in vascular cells. After incubation of vascular smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) with [14C]arachidonic acid, the profile of prostanoid synthesis was assessed by HPLC. Untransformed PGH2 released by the cells was evaluated as the difference in the formation of PGF2&agr; in the incubations performed in the presence and in the absence of SnCl2. Resting SMCs and SMCs stimulated with phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), interleukin (IL)-1&bgr;, and tumor necrosis factor (TNF)-&agr; formed PGE2 and PGI2 (evaluated as 6-oxo-PGF1&agr;), and in the presence of SnCl2 only a small amount of PGE2 was deviated toward PGF2&agr;. In contrast, resting and stimulated HUVECs produced PGI2, PGE2, PGF2&agr;, and PGD2, and SnCl2 completely diverted PGE2 and PGD2 toward PGF2&agr;. Reverse transcriptase–polymerase chain reaction analysis shows that mRNA encoding for PGES was not present in HUVECs and in endothelial cells from saphenous vein. Nevertheless, PGES was expressed in SMCs and induced by IL-1&bgr; and TNF-&agr;, and by PMA and LPS, although to a lesser extent. Whereas SMC stimulation led to an increase in the synthesis of PGE2 and PGI2 but not of untransformed PGH2, stimulation of endothelial cells resulted in an enhanced release of the vasoconstricting prostanoid PGH2.

Peroxisome-proliferator-activated receptor α agonists inhibit cyclo-oxygenase 2 and vascular endothelial growth factor transcriptional activation in human colorectal carcinoma cells via inhibition of activator protein-1

Recent evidence indicates that PPAR (peroxisome-proliferator-activated receptor) alpha ligands possess anti-inflammatory and antitumoural properties owing to their inhibitory effects on the expression of genes that are involved in the inflammatory response. However, the precise molecular mechanisms underlying these effects are poorly understood. In the present study, we show that tumour promoter PMA-mediated induction of genes that are significantly associated with inflammation, tumour growth and metastasis, such as COX-2 (cyclo-oxygenase 2) and VEGF (vascular endothelial growth factor), is inhibited by PPARalpha ligands in the human colorectal carcinoma cell line SW620. PPARalpha activators LY-171883 and WY-14,643 were able to diminish transcriptional induction of COX-2 and VEGF by inhibiting AP-1 (activator protein-1)-mediated transcriptional activation induced by PMA or by c-Jun overexpression. The actions of these ligands on AP-1 activation and COX-2 and VEGF transcriptional induction were found to be dependent on PPARalpha expression. Our studies demonstrate the existence of a negative cross-talk between the PPARalpha- and AP-1-dependent signalling pathways in these cells. PPARalpha interfered with at least two steps within the pathway leading to AP-1 activation. First, PPARalpha activation impaired AP-1 binding to a consensus DNA sequence. Secondly, PPARalpha ligands inhibited c-Jun transactivating activity. Taken together, these findings provide new insight into the anti-inflammatory and anti-tumoural properties of PPARalpha activation, through the inhibition of the induction of AP-1-dependent genes that are involved in inflammation and tumour progression.

Involvement of PGE2 and the cAMP signalling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS-activated macrophages

PG (prostaglandin) E2 plays an important role in the modulation of the immune response and the inflammatory process. In the present study, we describe a PGE2 positive feedback for COX (cyclo-oxygenase)-2 and mPGES-1 [microsomal PGES (PGE synthase)-1] expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dbcAMP (dibutyryl-cAMP) or forskolin. Furthermore, the cAMP signalling pathway co-operates with LPS (lipopolysaccharide) in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of PGE receptors [EPs (E-prostanoids)] showed that incubation with EP2 agonists up-regulated both COX2 and mPGES-1 mRNA levels. Moreover, EP2 receptor overexpression enhanced the transcriptional activation of COX2 and mPGES-1 promoters. This induction was repressed by the PKA (protein kinase A) inhibitor H89. Activation of the PGE2/EP2/PKA signalling pathway induced the phosphorylation of CREB [CRE (cAMP-response element)-binding protein] in macrophages and stimulated the specific binding of this transcription factor to COX2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, the results of the present study demonstrate that activation of PKA/CREB signalling through the EP2 receptor by PGE2 plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

ERK5 Activates NF-κB in Leukemic T Cells and Is Essential for Their Growth In Vivo

MAPK cascades play a central role in the cellular response to the environment. The pathway involving the MAPK ERK5 mediates growth factor- and stress-induced intracellular signaling that controls proliferation or survival depending upon the cell context. In this study, we show that reducing ERK5 levels with a specific small hairpin RNA 5 (shERK5) reduced cell viability, sensitized cells to death receptor-induced apoptosis, and blocked the palliative effects of phorbol ester in anti-Fas Ab-treated cells. shERK5 decreased nuclear accumulation of the NF-κB p65 subunit, and conversely, ectopic activation of ERK5 led to constitutive nuclear localization of p65 and increased its ability to trans activate specific reporter genes. Finally, the T lymphoma cell line EL-4, upon expression of shERK5, proliferated in vitro, but failed to induce s.c. tumors in mice. Our results suggest that ERK5 is essential for survival of leukemic T cells in vivo, and thus represents a promising target for therapeutic intervention in this type of malignancy.

Characterization of the promoter region and flanking sequences of the neuron-specific gene RC3 (neurogranin)

RC3 encodes a thyroid hormone-dependent, calmodulin-binding, protein kinase C substrate (neurogranin, p17) present in the dendritic spines of discrete neuronal populations in the forebrain. Its physiological role could be related to synaptic plasticity, memory, and other processes. In the present work we have isolated and sequenced 2.4 kbp of genomic DNA upstream from the origin of transcription and determined its nucleotide sequence. The major features of the RC3 promoter are the absence of TATA and CAAT boxes and the presence of an Initiator sequence surrounding the cap site. By sequence analysis we identified several cis-acting regulatory elements, among them response elements for retinoic acid and steroid (glucocorticoids/progesterone) hormone receptors. An oligonucleotide containing the retinoic acid responsive element bound to retinoic acid receptors specifically in vitro and conferred retinoic acid regulation to a heterologous promoter after transfection in COS-7 cells. Retinoic acid and dexamethasone, respectively, increased activity of the RC3 promoter in neuroblastoma cells when a deletion construct containing the retinoic acid and the glucocorticoid responsive elements was cotransfected with retinoic acid receptor or glucocorticoid receptor expression vectors. When added together all-trans retinoic acid and dexamethasone had additive effects. Despite the fact that RC3 expression in vivo is thyroid hormone-dependent, no evidence for the presence of a thyroid hormone responsive element was found within the 2.4 kbp flanking region analyzed and thyroid hormone did not increase reporter activity after cotransfection of suitable constructs with thyroid hormone receptor expression vectors. Our results suggest that the expression of RC3 in vivo could be subject to complex physiological signals, including retinoids and steroid hormones in addition to thyroid hormones.

Coordinated up-regulation of cyclooxygenase-2 and microsomal prostaglandin E synthase 1 transcription by nuclear factor kappa B and early growth response-1 in macrophages.

Prostaglandin (PG) E(2) is a potent lipid mediator that plays an essential role in inflammation, fever and pain. It is produced from arachidonic acid (AA) by a cascade of enzymatic reactions involving cyclooxygenases (COX-1 and -2) and prostaglandin E synthases (cPGES, mPGES-1 and -2). Functional coupling of the inducible enzymes COX-2 and mPGES-1 has been proposed for increased production of PGE(2) in different cell types. PGE(2) produced by macrophages plays an essential role in the pathogenesis of inflammatory diseases. Here, we have investigated the mechanisms involved in the regulation of COX-2 and mPGES-1 expressions in murine macrophages upon bacterial lipopolysaccharide (LPS) treatment. LPS stimulation induced the coordinated synthesis of COX-2 and mPGES-1 that resulted in an enhanced production of PGE(2) in RAW 264.7 macrophages. Furthermore, we show the involvement of NF-kappaB and Egr-1 transcription factors in the transcriptional induction of these enzymes. LPS treatment promoted specific binding of NF-kappaB to both COX-2 and mPGES-1 promoters. Site-directed mutagenesis, electrophoretic mobility shift assays and ChIP assays allowed the identification of a sequence acting as a NF-kappaB recognition site in the murine mPGES-1 promoter. Furthermore, LPS induced the expression of Egr-1 that cooperated with NF-kappaB in the up-regulation of COX-2 and mPGES-1. Inhibition of Egr-1 expression reduced substantially LPS-mediated induction of COX-2 and mPGES-1 expression, resulting in a decrease in PGE(2) production. Our findings point out to Egr-1 and NF-kappaB cooperation as determinant for PGE2 synthesis by macrophages in inflammatory processes through the coordinated regulation of COX-2 and mPGES-1.