Paola Scodelaro Bilbao

ATP stimulates the proliferation of MCF-7 cells through the PI3K/Akt signaling pathway.

We studied the modulation of the PI3K/Akt signaling pathway by ATP in MCF-7 cells. Western blot analysis showed that ATP stimulated the phosphorylation of Akt in a dose- and time-dependent manner. Akt phosphorylation in response to nucleotides followed the potency order ATP=UTP=ATPgammaS>>ADP=UDP>ADPbetaS=adenosine, suggesting participation of P2Y(2/4) receptors. Inhibitors of PI3K, PLC, PKC and Src or Src antisense oligonucleotides prevented ATP-induced phosphorylation of Akt. Incubation of cells with 2-APB or in a nominally Ca(2+)-free medium plus EGTA showed that Akt phosphorylation by ATP depends on intracellular calcium release but is independent of calcium influx. The PI3K inhibitor was not effective in reducing MAPKs phosphorylation by ATP. ATP and UTP stimulated MCF-7 cell proliferation, effect that was inhibited by PI3K, PLC, PKC, Src and MAPKs inhibitors. These findings suggest that ATP modulation of P2Y(2/4) receptors increases MCF-7 cell proliferation by activation of the PI3K/Akt signaling pathway through PLC/IP(3)/Ca(2+), PKC and Src.

Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line.

BACKGROUND ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell. METHODS AND RESULTS We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 microM ATP. Moreover, ATPgammaS, UTP, and UDP but not ADP or ADPbetaS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 and P2Y6 receptor subtypes. RT-PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca2+ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR. GENERAL SIGNIFICANCE These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells.

Protein phosphatases: Possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K(d)=1.39 ± 0.33 μM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K(d)=1.42 ± 0.15 μM, 2.00 ± 0.2 μM and 2.4 ± 0.4 μM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD. As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na₃VO₄ and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.

ATP modulates transcription factors through P2Y2 and P2Y4 receptors via PKC/MAPKs and PKC/Src pathways in MCF-7 cells

In this work, we studied the involvement of PKC and Src in the phosphorylation of ERK1/2, p38 and JNK1 MAPKs and in the modulation of ATF-1, c-Fos, c-Jun and Jun D transcription factors by ATP in MCF-7 breast cancer cells. RT-PCR studies and nucleotide sequence analysis confirmed first the expression of P2Y(2)- and P2Y(4)-receptor subtypes. The use of specific inhibitors and Src antisense oligonucleotides showed that PKC, but not Src, plays a role in the phosphorylation of MAPKs by ATP. ATP stimulated the expression of c-Fos and the phosphorylation c-Jun, Jun D and ATF-1. PKC and Src only participated in c-Fos induction and in ATF-1 phosphorylation. Pharmacological inhibition of MAPKs demonstrated that c-Fos induction and phosphorylation of c-Jun and Jun D, but not of ATF-1, depend on MAPK activation. These results suggest that stimulation of P2Y(2) and P2Y(4) receptors by ATP modulates transcription factors through PKC/MAPKs and PKC/Src pathways in MCF-7 cells.

Interaction of purinergic receptors with GPCRs, ion channels, tyrosine kinase and steroid hormone receptors orchestrates cell function

Extracellular purines and pyrimidines have emerged as key regulators of a wide range of physiological and pathophysiological cellular processes acting through P1 and P2 cell surface receptors. Increasing evidence suggests that purinergic receptors can interact with and/or modulate the activity of other classes of receptors and ion channels. This review will focus on the interactions of purinergic receptors with other GPCRs, ion channels, receptor tyrosine kinases, and steroid hormone receptors. Also, the signal transduction pathways regulated by these complexes and their new functional properties are discussed.

Extracellular ATP regulates FoxO family of transcription factors and cell cycle progression through PI3K/Akt in MCF-7 cells.

BACKGROUND Forkhead Box-O (FoxO) transcription factors regulate the expression of many genes involved in suppression. Released nucleotides can regulate intracellular signaling pathways through membrane-bound purinergic receptors, to promote or prevent malignant cell transformation. We studied the role of extracellular ATP in the modulation of Forkhead Box O (FoxO) transcription factors and of cell cycle progression in MCF-7 breast cancer cells. METHODS Western blot analysis, cell transfections with siRNA against Akt, immunocytochemistry, subcellular fractionation studies and flow cytometry analysis were performed. RESULTS ATP induced the phosphorylation of FoxO1/3a at threonine 24/32, whereas reduced the expression of FoxO1. In addition, ATP increased the expression of the cyclins D1 and D3 and down-regulated the cell cycle inhibitory proteins p21Cip1 and p27Kip1. The use of the phosphatidylinositol 3 kinase (PI3K) inhibitor, Ly294002, and/or of siRNA to reduce the expression of the serine/threonine kinase Akt showed that these effects are mediated by the PI3K/Akt signaling pathway. ATP induced the translocation of FoxO3a from the nucleus to the cytoplasm. Also, ATP increased the number of cells in the S phase of cell cycle; this effect was reverted by the use of Ly294002 and the proteasome inhibitor bortezomib. CONCLUSION Extracellular ATP induces the inactivation of FoxO transcription factors and cell cycle progression through the PI3K/Akt pathway in MCF-7 cells. GENERAL SIGNIFICANCE These findings provide new molecular basis for further understanding the mechanisms involved in ATP signal transduction in breast cancer cells, and should be considered for the development of effective breast cancer therapeutic strategies.

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.

Fatty Acids from Microalgae: Targeting the Accumulation of Triacylglycerides

Microalgae were originally considered as sources of long-chain polyunsaturated fatty acids (PUFAs), mainly for aquaculture purposes. However, based on the fact that their fatty acids (FA), stored as triacylglycerides (TAG), can be converted into biodiesel via a transesterification reaction, several microalgal species have emerged over the last decade as promising feedstocks for biofuel production. Elucidation of microalgae FA and TAG metabolic pathways is therefore becoming a cutting-edge field for developing transgenic algal strains with improved lipid accumulation ability. Furthermore, many of the biomolecules produced by microalgae can also be exploited. In this chapter, we describe recent advances in the field of FA and TAG pathways in microalgae, focusing in particular on the enzymes involved in FA and TAG synthesis, their accumulation in lipid droplets, and their degradation. Mention is made of potentially high-value products that can be obtained from microalgae, and possible molecular targets for enhancing FA and TAG production are outlined. A summary is provided of transcriptomics, proteomics, and metabolomics of the above-mentioned pathways in microalgae. Understanding the relation between anabolic and catabolic lipid enzyme pathways will provide new insights into biodiesel production and other valuable biomolecules obtained from microalgae.