Francisco Javier de Lucio-Cazaña

All-trans retinoic acid induces COX-2 and prostaglandin E2 synthesis in SH-SY5Y human neuroblastoma cells: involvement of retinoic acid receptors and extracellular-regulated kinase 1/2

BackgroundOur recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity.MethodsCultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test.ResultsATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-β expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells.ConclusionThese results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.

Mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells

Hypoxia-inducible factor-1α (HIF-1α) and all-trans retinoic acid (ATRA) afford protection in several experimental models of kidney disease. HIF-1α protein is degraded under normoxia but stabilized by hypoxia, which activates its transcription factor function. ATRA activates another set of transcription factors, the retinoic acid receptors (RAR) α, β and γ, which mediate its effects on target genes. ATRA also up-regulates the expression of RAR α, β and γ at the transcriptional level. Here we demonstrate the presence of mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells. In human proximal tubular HK-2 cells we have found that: (i) ATRA treatment induces HIF-1α under normoxic conditions and also synergizes with hypoxia leading to the over-expression of HIF-1α and vascular endothelial growth factor-A, a HIF-1α-regulated renal protector. ATRA-induced HIF-1α expression involved stabilization of HIF-1α mRNA but not of HIF-1α protein. (ii) Expression of HIF-1α is an absolute requirement for the transcriptional up-regulation of RARβ by ATRA. Transfection with HIF-1α siRNA abolished the induction by ATRA of the expression of both RARβ mRNA and protein while treatment with HIF-1α inhibitor YC-1 results in the abolishment of ATRA-induced activity of a retinoic acid-response element (RARE) construct from the RARβ promoter. (iii) Hypoxia up-regulates RARβ through HIF-1α since this effect was inhibited by HIF-1α knockdown. In contrast to ATRA-induced RARβ up-regulation, induction of RARβ expression by ATRA did not involve transcriptional up-regulation as hypoxia did not increase the expression of RARβ mRNA or the activity of the RARE construct. These results suggest the presence of crosstalk between hypoxia/HIF-1α and ATRA/RARβ that may be physiologically and pharmacologically relevant.

Intracrine prostaglandin E2 signalling regulates hypoxia-inducible factor-1α expression through retinoic acid receptor-β

We have previously found in human renal proximal tubular HK-2 cells that hypoxia- and all-trans retinoic acid-induced hypoxia-inducible factor-1α up-regulation is accompanied by retinoic acid receptor-β up-regulation. Here we first investigated whether hypoxia-inducible factor-1α expression is dependent on retinoic acid receptor-β and our results confirmed it since (i) hypoxia-inducible factor-1α-inducing agents hypoxia, hypoxia-mimetic agent desferrioxamine, all-trans retinoic acid and interleukin-1β increased retinoic acid receptor-β expression, (ii) hypoxia-inducible factor-1α up-regulation was prevented by retinoic acid receptor-β antagonist LE-135 or siRNA retinoic acid receptor-β and (iii) there was direct binding of retinoic acid receptor-β to the retinoic acid response element in hypoxia-inducible factor-1α promoter upon treatment with all-trans retinoic acid and 16,16-dimethyl-prostaglandin E(2). Since intracellular prostaglandin E(2) mediates hypoxia-inducible factor-1α up-regulation in normoxia in HK-2 cells, we next investigated and confirmed, its role in the up-regulation of retinoic acid receptor-β in normoxia by hypoxia-inducible factor-1α-inducing agents all-trans retinoic acid, interleukin-1β and 16,16-dimethyl-prostaglandin E(2) by inhibiting cyclooxygenases, prostaglandin influx transporter or EP receptors. Interestingly, the hypoxia-induced increase in retinoic acid receptor-β expression and accumulation of hypoxia-inducible factor-1α was also blocked by the inhibitors tested. This is the first time, to our knowledge, that retinoic acid receptor-β signalling is involved in the control of the expression of transcription factor hypoxia-inducible factor-1α in both normoxia and hypoxia and that retinoic acid receptor-β expression is found to be strictly regulated by intracellular prostaglandin E(2). Given the relevance of hypoxia-inducible factor-1α in the kidney in terms of tumorigenesis, progressive renal failure, production of erythropoietin and protection in several models of renal disease, our results open new therapeutic opportunities on the control of hypoxia-inducible factor-1α based upon the pharmacological modulation of retinoic acid receptor-β, either directly or through the control of intracellular prostaglandin E(2) levels/signalling.

Kinase‐dependent, retinoic acid receptor‐independent up‐regulation of cyclooxygenase‐2 by all‐trans retinoic acid in human mesangial cells

Preliminary results in human mesangial cells (MC) suggested that all‐trans retinoic acid (ATRA) increased the expression of COX‐2 and the production of prostaglandin E2 (PGE2), a PG with anti‐inflammatory effects in MC. The aim of this work is to confirm that ATRA increases the expression of COX‐2 in MC and to examine the mechanisms involved.

Upregulation of Cyclooxygenases by Retinoic Acid in Rat Mesangial Cells

All-trans retinoic acid (ATRA) increases the expression of COX-1 and COX-2 and the production of PGE2, a prostaglandin with anti-inflammatory effects in human mesangial cells (MC). COX-2 increased through a transcriptional mechanism independent of retinoic acid receptors (RAR) and retinoid X receptors (RXR) and dependent on extracellular regulated kinase-1/2 (ERK1/2), that became phosphorylated 5 min after ATRA addition. Here, in rat MC, ATRA also upregulated COX isoenzymes and PGE2 production, but not in the same way as in human MC: (1) PGE2 production increased only slightly; (2) RAR and RXR were involved in the transcriptional upregulation of COX-2 by ATRA since the RAR-pan-antagonist AGN193109 or the RXR-pan-antagonist HX531 abolished the induction of COX-2 mRNA whereas the RAR-pan-agonist TTNPB or the RXR-pan-agonist AGN194204 induced expression of COX-2, and (3) ERK1/2 phosphorylation, though important for COX-2 upregulation, took more than 1 h. Therefore the regulation of COX by ATRA exhibits striking differences between human and rat MC.

Transactivation of EGFR by prostaglandin E2 receptors: a nuclear story?

The pharmacological modulation of hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. Although it is assumed that prostaglandin E2 (PGE2) exerts its biological effects from the extracellular medium through activation of EP receptors located at the cell membrane, we have shown in human renal proximal tubular HK-2 cells (and other cell lines) that intracellular PGE2 regulates the expression of HIF-1α expression and the production of VEGF-A. Here, we have found—through experiments involving EP receptors agonists, EP receptor gene silencing and inhibition of the prostaglandin uptake transporter—that these biological effects of PGE2 are mediated by intracellular EP2 receptors. In sharp contrast with cell membrane EP2, whose activation results in increased production of cAMP, intracellular EP2 signaling was independent of cAMP. Instead, it involved c-src-dependent transactivation of epidermal growth factor receptor, which led to p38/ERK1/2-dependent activation of mitogen- and stress-activated kinase-1 (MSK-1) and to MSK-1-dependent-histone H3 phosphorylation and transcriptional up-regulation of retinoic acid receptor-β. Even more important, this signaling pathway was fully reproduced in nuclei isolated from HK-2 cell, which highlights the relevance of nuclear EP receptors in the up-regulation of HIF-1α. These results open the possibility that signal cascades that proceed entirely in the cell nucleus might be responsible for several PGE2 effects that are assumed to be due to cell membrane EP receptors.

Microparticles released by vascular endothelial cells increase hypoxia inducible factor expression in human proximal tubular HK-2 cells.

Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases.

All-Trans Retinoic Acid and Glycated Albumin Reciprocally Influence their Effects in Human Mesangial Cells

UNLABELLED All-trans-retinoic acid (tRA) modulates in human mesangial cells (MC) antioxidant defenses, the expression of interleukin-1beta-induced vascular cell-adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and the retinoic acid-receptor-beta (RAR-beta). The correlation of the serum levels of glycated hemoglobin A1c with tRA in type 2 diabetes mellitus patients led us to hypothesize that tRA and glycated albumin (GA), the main circulating glycated protein, might mutually interact in MC. We studied 1) the influence of tRA on GA effects in cultured MC: an assessment was made on how pre-incubation with tRA modified the effects of GA on intracellular oxidation and on the expression of mRNA and protein of COX-2 and VCAM-1; and 2) the influence of GA on tRA effects in MC: we studied how the induction of RARbeta expression by tRA was modified by GA. RESULTS GA dose-dependently increased intracellular oxidation and the expression of the molecules involved in leukocyte infiltration, namely COX-2 and VCAM-1 . Pre-incubation with tRA exacerbated GA effects by up to a three- to four-fold additional increase. In turn, induction by tRA of RAR-beta was fully inhibited by GA. Thus tRA and GA reciprocally influence their effects in MC. It is possible that this interaction may have a pathophysiological or pharmacological role in diabetic nephropathy.

The effect of the European traditional use directive on the register of herbal medicinal products in Spain

BACKGROUND Directive 2004/24/EC, which came into force in 2011, created new regulatory requirements for traditional herbal medicines (THM). This study compared the Spanish THM registry before and after the Directive came fully into force in 2011. METHODS We consulted the herbal medicinal plant and drug catalogues (General Council of the Official Colleges of Pharmacists), the website of the European Medicines Agency (EMA), and retail web sites. RESULTS Of 315 THM (from 39 companies) licensed in Spain in 2010, only 48 (10 companies) remained licensed in 2013, mainly due to their withdrawal: the EMA had received just 123 applications from Spain and at least 34% formerly licensed THM had shifted to the less strictly regulated food sector, while up to 54% might have disappeared from the market. However, there is still a significant presence of retail websites making illegal health claims. CONCLUSION In Spain, the public health benefits of the Directive 2004/24/EC might be less than expected.

PROSTAGLANDIN E2 stimulates cancer-related phenotypes in prostate cancer PC3 cells through cyclooxygenase-2: MADRIGAL-MARTÍNEZ et al.

Cyclooxygenase (COX)‐derived prostaglandin E2 (PGE2) affects many mechanisms that have been shown to play roles in carcinogenesis. Recently, we found that, in androgen‐independent prostate cancer PC3 cells, PGE2 acts through an intracrine mechanism by which its uptake by the prostaglandin transporter (PGT) results in increased intracellular PGE2 (iPGE2), leading to enhanced cell proliferation, migration, invasion, angiogenesis, and loss of cell adhesion to collagen I. These iPGE2‐mediated effects were dependent on hypoxia‐inducible factor 1‐α (HIF‐1α), whose expression increased upon epidermal growth factor receptor (EGFR) transactivation by a subset of intracellular PGE2 receptors. Here, we aimed to study the role of COX in PGE2 protumoral effects in PC3 cells and found that the effects were prevented by inhibition of COX‐2, which highlights its crucial role amplifying the levels of iPGE2. Treatment with exogenous PGE2 determined a transcriptional increase in COX‐2 expression, which was abolished by genetic or pharmacologic inhibition of PGT. PGE2‐induced increase in COX‐2 expression and, thereby, in transcriptional increase in HIF‐1α expression was due to EGFR activation, leading to the activation of Phosphoinositide 3‐kinase/Akt, Extracellular signal ‐regulated kinases 1/2, p38 and Mitogen‐ and stress‐activated protein kinase‐1 (PI3K/Akt, Erk1/2, p38 and MSK‐1). Collectively, the data suggest that EGFR‐dependent COX‐2 upregulation by a novel positive feedback loop triggered by iPGE2 underlies the intracrine pro‐tumoral effects of PGE2 in PC3 cells. Therefore, this feedback loop may be relevant in prostate cancer for the maintenance of PGE2‐dependent cancer cell growth through amplifying the activity of the COX‐2 pathway.