Eugenio Jiménez

Activation of MAP Kinase by Muscarinic Cholinergic Receptors Induces Cell Proliferation and Protein Synthesis in Human Breast Cancer Cells

Carbachol (Cch), a muscarinic acetylcholine receptor (mAChR) agonist, increases intracellular‐free Ca2+ mobilization and induces mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) phosphorylation in MCF‐7 human breast cancer cells. Pretreatment of cells with the selective phospholipase C (PLC) inhibitor U73122, or incubation of cells in a Ca2+‐free medium did not alter Cch‐stimulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by phorbol 12‐myristate acetate (PMA), an activator of protein kinase C (PKC), but Cch‐evoked MAPK/ERK activation was unaffected by down‐regulation of PKC or by pretreatment of cells with GF109203X, a PKC inhibitor. However, Cch‐stimulated MAPK/ERK phosphorylation was completely blocked by myristoylated PKC‐ζ pseudosubstrate, a specific inhibitor of PKC‐ζ, and high doses of staurosporine. Pretreatment of human breast cancer cells with wortmannin or LY294002, selective inhibitors of phosphoinositide 3‐kinase (PI3K), diminished Cch‐mediated MAPK/ERK phosphorylation. Similar results were observed when MCF‐7 cells were pretreated with genistein, a non‐selective inhibitor of tyrosine kinases, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF‐7 human breast cancer cells mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen activated kinase kinase. In conclusion, analyses of mAChR downstream effectors reveal that PKC‐ζ, PI3K, and Src family of tyrosine kinases, but not intracellular‐free Ca2+ mobilization or conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK activation. In addition, MAPK/ERK are involved in the regulation of growth and proliferation of MCF‐7 human breast cancer cells.

P2Y receptors activate MAPK/ERK through a pathway involving PI3K/PDK1/PKC-zeta in human vein endothelial cells.

Aims: In this study we investigated the effects of P2 receptors in the regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in human umbilical vein endothelial cells (HUVEC). Methods: Cytosolic Ca2+ concentration ([Ca2+]i) was measured using fura-2/AM, and MAPK/ ERK phosphorylation using Western blot analysis. Results: ATP, 2-meSATP, UTP and UDP cause a rapid and transitory increase in the phosphorylation of MAPK/ERK. In contrast, negligible response was seen for a,ß-meATP, a general P2X receptors agonist. ATPdependent activation of MAPK/ERK was prevented by pretreatment of HUVEC with pertussis toxin or MEK inhibitor PD98059. In addition, activation of the MAPK/ ERK cascade by ATP was blocked in cells pretreated with wortmannin and LY294002, but not by U73122, BAPTA or a Ca2+-free medium. Furthermore, an inhibition of ATP-dependent MAPK/ERK phosphorylation was observed in HUVEC pretreated with high doses of GF109203X or myristoylated PKC- ζ pseudosubstrate. Similar results were observed when cells were pretreated with the Src tyrosine kinase inhibitor PP2. However, ATP-stimulated MAPK/ERK activation was unaffected in cells pretreated with AG1478 or perillic acid. We also found that ATP stimulates both the phosphorylation of 3- phosphoinositide-dependent protein kinase-1 (PDK1) and its translocation to plasma membrane in a timedependent manner. Conclusion: These observations suggest that the effects mediated by ATP in HUVEC occur via PTX-sensitive G-protein-coupled P2Y receptors through PI3K-dependent mechanisms, in which PDK1 and PKC-ζ are two key molecules within signal cascade leading to MAPK/ERK activation.

Angiotensin II induces MMP 2 activity via FAK/JNK pathway in human endothelial cells.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of cardiovascular diseases and are modified in response to a variety of stimuli such as bioactive peptides, cytokines and/or grown factors. In this study, we demonstrated that angiotensin II (Ang II) induces a time- and dose-dependent increase in the activity of metalloproteinase 2 (MMP 2) in human umbilical vein endothelial cells (HUVEC). The effect of Ang II was markedly attenuated in cells pretreated with wortmannin and LY294002, two selective inhibitors of phosphatidylinositol-3-kinase (PI3K), indicating that PI3K plays a key role in regulating MMP 2 activity. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific Src-family tyrosine kinase inhibitor PP2, demonstrating the involvement of protein tyrosine kinases, and particularly Src-family tyrosine kinases on the downstream signaling pathway of Ang II receptors. Furthermore, Ang II-induced MMP 2 activation was markedly blocked by SP600125, a selective c-Jun N-terminal kinase (JNK) inhibitor, or pre-treatment of cells with antisense oligonucleotide to focal adhesion kinase (FAK), indicating that both molecules were important for the activation of MMP 2 by Ang II receptor stimulation. In conclusion, these results suggest that Ang II mediates an increase in MMP 2 activity in macrovascular endothelial cells through signal transduction pathways dependent on PI3K and Src-family tyrosine kinases activation, as well as JNK and FAK phosphorylation.

The renin–angiotensin system in thyroid disorders and its role in cardiovascular and renal manifestations

Thyroid disorders are among the most common endocrine diseases and affect virtually all physiological systems, with an especially marked impact on cardiovascular and renal systems. This review summarizes the effects of thyroid hormones on the renin-angiotensin system (RAS) and the participation of the RAS in the cardiovascular and renal manifestations of thyroid disorders. Thyroid hormones are important regulators of cardiac and renal mass, vascular function, renal sodium handling, and consequently blood pressure (BP). The RAS acts globally to control cardiovascular and renal functions, while RAS components act systemically and locally in individual organs. Various authors have implicated the systemic and local RAS in the mediation of functional and structural changes in cardiovascular and renal tissues due to abnormal thyroid hormone levels. This review analyzes the influence of thyroid hormones on RAS components and discusses the role of the RAS in BP, cardiac mass, vascular function, and renal abnormalities in thyroid disorders.

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.

Cisplatin reduces endothelial cell migration via regulation of type 2-matrix metalloproteinase activity.

Aims: In this study we investigated the effect of cisplatin on endothelial cell migration, an essential process for vascular remodeling and regeneration in several physiological and pathological situations. Material and Methods: Human umbilical vein endothelial cells (HUVEC) were treated with cisplatin and endothelial cell migration analyzed by fluorescence and scratch-wound migration assay. MMP2 and MMP9 activity were determined by zymographic assay, and MAPK activation by Western blotting analysis. Results: We demonstrated that cisplatin provoked a time- and dose-dependent decrease of HUVEC migration; this effect was clearly independent from its well known cytotoxic activity. In addition, cisplatin markedly reduced MMP2 activity in both conditioned media and cell lysates, increased p38 MAPK and JNK phosphorylation, but did not affect ERK phosphorylation. Endothelial cell migration was attenuated by treatment of cells with GM6001, a non-specific inhibitor of MMPs, or by a selective anti-MMP2 antibody. However, treatment of cells with SB202190 or SP600125, inhibitors of p38 MAPK and JNK respectively, did not affect HUVEC migration. Conclusion: These results suggested that cisplatin induced a reduction of endothelial cell migration through an inhibition of MMP2 activity by downstream signal transduction pathways independent of JNK and p38 MAPK activation.

Clusterin expression is associated with decreased disease‐free survival of patients with colorectal carcinomas

Redondo M, Rodrigo I, Alcaide J, Tellez T, Roldan M J, Funez R, Diaz‐Martin A, Rueda A & Jiménez E
(2010) Histopathology 56, 932–936
Clusterin expression is associated with decreased disease‐free survival of patients with colorectal carcinomas

Prognostic implications of emergency admission and delays in patients with breast cancer

IntroductionThis study evaluates clinical–pathological characteristics and survival rates associated with emergency admission and delays in diagnosis and treatment of 411 consecutive breast cancer patients.Materials and methodsEmergency admission and first symptom–first hospital visit delay were significantly associated with advanced tumor stages but only in the former case with short disease-free survival (RR 2.5, CI 95% 1.5–4.2).ResultsBrief diagnostic delays were significantly associated with advanced disease stage and poor survival rates (RR 2.04; CI 95% 1.08–3.82) probably because sicker patients receive prompt medical attention.

Glucocorticoid receptors in the euryhaline teleost Anguilla anguilla.

To determine the importance of glucocorticoids in the salt water adaptation of European yellow eel we have evaluated the concentration, affinity and physical properties of glucocorticoid receptors (GR) in gill from both sea water- (SW) and freshwater-adapted (FW) animals. Using ligand binding techniques we demonstrated that high affinity GR were present in both cytosolic and nuclear fractions obtained from whole gill. Isoelectric focusing (IEF) of branchial GR indicated the presence of two distinct species, with pI values of 6.1 and 6.7. The form at pI 6.7 sedimented with a Svedberg constant of 4S on glycerol density gradients while the pI 6. 1 sedimented in fractions corresponding to 9S. Treatment of the pI 6. 1 form with urea (4 M) resulted in generation of the form with pI 6. 7. The evidence thus suggested that the oligomeric urea-sensitive form (pI 6.1) contained a form of GR which, as a monomer, focused at pI 6.7. IEF revealed the same concentrations of the pI 6.7 form in both SW and FW. However, there was significantly more (3-fold) pI 6. 1 isoform in FW than in SW, and this form decreased gradually during the course of seawater transfer. A transient increase of the nuclear-bound GR was also observed during SW adaptation. The balance between these forms could represent a dynamic parameter with important implications regarding GR function and gill responses to cortisol in salt water adaptation in teleosts.

Activation of second messenger-dependent protein kinases induces muscarinic acetylcholine receptor desensitization in rat thyroid epithelial cells.

Internalization and phosphorylation of G protein-coupled receptors (GPCR) are considered two important regulatory events of receptor signal transduction. In Fischer rat thyroid (FRT) epithelial cells, we have shown that muscarinic acetylcholine receptor (mAChR) stimulation induces intracellular Ca2+ mobilization via Ca2+ store release, capacitative Ca2+ entry and voltage-dependent Ca2+ channels activation. In the present study, the role of mAChR internalization and phosphorylation on receptor signalling pathway was examined by means of intracellular Ca2+ measurement in these cells. Exposure of FRT cells to carbachol (Cch), a mAChR agonist, resulted in a desensitization of receptor-mediated intracellular Ca2+ mobilization and induced the internalization of constitutively expressed mAChR in this cell type. Treatment of FRT cells with hypertonic sucrose, which markedly reduced agonist-receptor complex internalization, or phenylarsine oxide (PAO) diminished the Cch-induced intracellular Ca2+ response. Moreover, pretreatment of cells with phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC), completely abolished Cch-evoked Ca2+ mobilization, whereas it was significantly increased by the preincubation of cells with GF109203X, a selective inhibitor of PKC. We also found a marked decrease on Cch-stimulated Ca2+ mobilization in pretreated FRT cells with forskolin, an activator of protein kinase A (PKA), but the preincubation of cells with genistein, an inhibitor of protein tyrosine kinases, had no effect on Ca2+ mobilization induced by Cch. These findings seem to indicate that mAChR in FRT cells exhibit a desensitization, which may be mediated, at least in part, through activation of second messenger-dependent protein kinases and that receptor internalization could be necessary for signalling.