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A novel pathway of cell growth regulation mediated by a PLA₂-derived phosphoinositide metabolite

Stefania Mariggiò^*,1, Jordi Sebastià^*, Beatrice Maria Filippi^*, Cristiano Iurisci^*, Cinzia Volonté, Susanna Amadio, Valentina De Falco, Massimo Santoro and Daniela Corda^*,1

^* Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy;

Santa Lucia Foundation/CNR, Rome, Italy; and

Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università Federico II di Napoli c/o Istituto di Endocrinologia e Oncologia Sperimentale del CNR, Naples, Italy

¹Correspondence: Department of Cell Biology and Oncology Consorzio Mario Negri Sud, Via Nazionale 8, 66030 Santa Maria Imbaro, Chieti, Italy. E-mail: mariggio{at}negrisud.it or corda{at}negrisud.it

SPECIFIC AIMS

The phosphoinositides (PIs) are membrane bilayer components with important roles in the control of many cellular functions. This is exerted either directly by the differently phosphorylated PIs or via the inositol-containing metabolites of PIs hydrolysis. The glycerophosphoinositols are water-soluble phospholipase A₂ (PLA₂) metabolites that, when added exogenously to cells, can affect cellular functions including proliferation, actin cytoskeleton organization, and tumor cell invasion. Here we investigate receptor-mediated regulation of PLA₂ activity that leads to glycerophosphoinositol (GroPIns) formation. We delineate the enzymes and regulatory cascade of phosphatidylinositol (PtdIns) hydrolysis and define its physiological significance. These data demonstrate a novel regulatory cascade that specifically involves PLA₂IV and GroPIns formation.

PRINCIPAL FINDINGS

1. Receptor-stimulated hydrolysis of membrane phosphoinositides through activation of PLA₂IV leads to increases in intracellular glycerophosphoinositol
In a continuous line of follicular thyroid cells (PCCl₃ cells) particularly suitable for studying control of cell proliferation and differentiation in an epithelial cell setting, GroPIns levels are increased by the addition of calcium ionophore (1 µM A23187), 10 µM ATP, and 20 µM L-noradrenaline hydrochloride (NE) (Fig. 1 A). These effects are concentration (1–100 µM) and time dependent. Other agonists (10^–7 M TSH, 1 µM carbachol) did not modulate GroPIns levels. In parallel, ATP and NE increased lysophosphatidylinositol (LysoPtdIns), the first product of PtdIns deacylation in cells (Fig. 1C ), and decreased PtdIns.

View larger version (13K): [in this window] [in a new window]   Figure 1. Agonist-induced production of GroPIns is mediated by PLA2IV in PCCl3 cells. Cells preincubated for 15 min without (–) and with (+) pyrrophenone (Pyr; 0.1 µM) were stimulated with ATP (10 µM) and NE (20 µM) for another 15 min unless otherwise specified. A) Stimulation of [3H]-GroPIns production (means±SE; 4 independent experiments). B) Time courses showing pyrrophenone effects on control and NE-stimulated [3H]-GroPIns levels (% of initial control levels). C) Stimulation of [3H]-LysoPtdIns production (means±SE; 4 independent experiments). D) As for panel B for [3H]-LysoPtdIns. E) Stimulation of [3H]-arachidonic acid release (% of untreated Ctrl cells; means±SE; 3 independent experiments). F) ATP- and NE-mediated [3H]-arachidonic acid release with increasing concentrations of pyrrophenone (% of untreated cells). *Significantly different from respective controls (P0.05).

ATP- and NE-induced increases in GroPIns were not prevented by inhibitors of Ca²⁺-independent PLA₂ (10 µM palmitoyl trifluoromethyl ketone, 1 µM bromo-enol lactone) or a secretory sPLA₂IIA inhibitor (50 µM sPLA₂IIA inhibitor-I). However, chelation of intracellular Ca²⁺ (10 µM BAPTA-AM) completely inhibited agonist-induced GroPIns formation; thus, the PLA₂ here is Ca²⁺ dependent. Finally, a specific inhibitor of cytosolic, Ca²⁺-dependent PLA₂IV (0.1 µM pyrrophenone) completely blocked ATP- and NE-induced GroPIns and LysoPtdIns formation (Fig. 1A –D).

In intact cells, PLA₂IV is selective for sn-2 arachidonic acid phospholipids. Here, ATP- and NE-induced GroPIns and LysoPtdIns formation are accompanied by release of this fatty acid (Fig. 1E ).

The ERK1/2 MAP and p38 stress activated protein kinases are regulators of PLA₂IV. Indeed, ATP- and NE-induced GroPIns formation was partially blocked (56–90%) by inhibitors of ERK1/2 and p38. Accordingly, these agonists induced increases in ERK1/2 and p38 phosphorylation, and consequent phosphorylation of PLA₂IV.

The 50% of PLA₂IV phosphorylated under normal growth conditions increased to 100% after ATP and NE stimulation (within 5 min and sustained for up to 3 h). The inhibitor U0126 completely prevented ATP- and NE-induced ERK activation and blocked the consequent PLA₂IV phosphorylation.

Thus, in PCCl₃ cells ATP and NE stimulate signaling pathways for hydrolysis of the PIs, with production of LysoPtdIns and GroPIns (and GroPIns4P) via activation of cytosolic, calcium-dependent PLA₂IV.

2. Correlation between PLA₂IV expression and GroPIns formation
To further substantiate the involvement of PLA₂IV, we produced permanently transfected PCCl₃ clones expressing empty vector (Vclone), wild-type (WT) (PLA₂IV_wt; WTclone), and dominant-negative (PLA₂IV_1–522; DNclone) PLA₂IV. In the WTclone, ATP-induced GroPIns and LysoPtdIns formation increased to 363% and 238% vs. Vclone; this was completely blocked by pyrrophenone. The DNclone showed 43% inhibition of ATP-induced GroPIns synthesis, with no effects on LysoPtdIns production. An siRNA specific for PLA₂IV promoted an 40% decrease in PLA₂IV and inhibited basal GroPIns by 25% and its ATP and NE stimulation by 30%.

In in vitro assays, PLA₂IV showed both phospholipase and lysolipase activities, hydrolyzing PtdIns to LysoPtdIns and LysoPtdIns to GroPIns, respectively. Collectively, these results further confirm PLA₂IV involvement in the formation and modulation of GroPIns levels in PCCl₃ cells.

3. PLA₂IV product involvement in regulation of PCCl₃ cell growth and differentiation
We investigated whether this purinergic and adrenergic receptor activation of PLA₂IV, and subsequent formation of these PIs metabolites, can mediate PCCl₃ cell differentiated functions. Although NE modestly affected thyroid differentiation (down-regulating thyroglobulin, PAX8, and TTF1 mRNA), the PLA₂IV pathway did not impair expression of these thyroid differentiation markers. Rather, GroPIns partially antagonized this effect.

For receptor modulation of cell proliferation, we investigated [³H]-thymidine incorporation in PCCl₃ cells in the absence of TSH, the main growth factor for PCCl₃ cells; proliferation was increased by 100 µM ATP and 20 µM NE (Fig. 2 A). This was completely prevented by pyrrophenone pretreatment, suggesting the involvement of a PLA₂IV-derived PIs metabolite in this modulation (Fig. 2A ).

View larger version (20K): [in this window] [in a new window]   Figure 2. PCCl3 cell growth is regulated by agonist stimulation of PLA2IV and PLA2IV-derived products. A) [3H]-Thymidine incorporation in cells stimulated for 48 h with ATP (100 µM) and NE (20 µM) without and with pyrrophenone (0.5 µM), GroPIns (100 µM, 400 µM), LysoPdtIns (10 µM), arachidonic acid (AA, 1 µM), and TSH (10–10 M) (means±SE; 5 independent experiments). **Significantly different from respective controls (P0.01). B) Representative growth curves of stable Vclone (empty vector), WTclone (PLA2IVwt), and DNclone (PLA2IV1–552) PCCl3 transfectants (means±SE; single experiment in triplicate of 3 independent experiments). C) Cell growth of WTclone in the absence (–) and presence (+) of pyrrophenone (0.1 µM) (means±SE; 3 independent experiments).

Exogenous addition of arachidonic acid (100 nM; 1 µM) also induced increases in [³H]-thymidine incorporation (Fig. 2A ), although involvement of its metabolites (PGE₁, PGE₂, and TXB₂; 10–300 nM) was excluded, as they and the general cyclooxygenase inhibitor lysinate acetylsalicylic acid (300 µM) had no effect on cell growth stimulation. Platelet-activating factor, another potentially active PLA₂ product, was ineffective (0.1–10 µM). Instead, exogenous addition of 100–400 µM GroPIns and 10 µM LysoPtdIns induced increases in [³H]-thymidine incorporation (Fig. 2A ).

The WTclone also showed a higher growth rate vs. the Vclone in medium without TSH, a difference abolished by pyrrophenone (Fig. 2B, C ), thus indicating that the higher growth rate is a consequence of PLA₂IV activity. In complete medium, TSH stimulation overcame this PLA₂IV effect. The Vclone and WTclone had similar growth rates; the DNclone did not grow without TSH (Fig. 2B ).

CONCLUSIONS AND SIGNIFICANCE

We have elucidated a novel regulatory cascade specifically linking receptor activation of PLA₂IV to regulation of cell proliferation through formation of the PtdIns metabolite GroPIns, adding to the regulatory pathways that are controlled by inositol-containing molecules.

The criteria applied to investigate coupling of the activated receptors to PLA₂IV activity were a requirement for Ca²⁺, use of inhibitors and RNA interference, and overexpression and down-regulation of PLA₂IV, allowing us to define this signaling cascade in these cells (Fig. 3 ). Thus, by stimulating PLC, activated purinergic and adrenergic receptors produce increases in [Ca²⁺]_i that promote activation of MAP kinases and PLA₂IV membrane translocation. ERK1/2 are activated downstream of this [Ca²⁺]_i increase, and phosphorylate and activate PLA₂IV (Fig. 3) . This agrees with activation of PLA₂IV reported in other cell systems and delineates the specific pathway leading to PLA₂IV-mediated PtdIns hydrolysis in PCCl₃ cells (Fig. 3) .

View larger version (82K): [in this window] [in a new window]   Figure 3. Schematic representation of the signaling cascade in the formation of GroPIns in PCCl3 cells. Pathways initiated by adrenergic (1, 2) and purinergic (P2Y2) receptor activation lead to PTX-sensitive activation of p38 and to PTX-insensitive activation of ERK1/2 (only for the adrenergic receptor) and PLC. These in turn either directly activate PLA2IV or produce an increase in [Ca2+]i; the latter promotes PLA2IV translocation to the membrane and ERKs activation. PLA2IV activation results in hydrolysis of PtdIns, release of arachidonic acid and LysoPtdIns, and finally, GroPIns production. See text for details.

We investigated whether this PtdIns hydrolysis mediates differentiated functions regulated by these ligands. In PCCl₃ cells, proliferation and differentiation controls are strictly coupled: TSH-regulated signals promote cell growth and differentiation while signals activated by oncogenes impair differentiation and stimulate TSH-independent proliferation. ATP and NE clearly stimulated PCCl₃ cell growth in a PLA₂IV-dependent manner, as their effects were inhibited by pyrrophenone and mimicked by PLA₂IV-derived metabolites GroPIns and LysoPtdIns. For GroPIns, the micromolar concentrations used agree well with cytosolic levels of GroPIns, implying it is a good candidate for modulation of adrenergic- and purinergic-receptor-mediated control of cell growth. Conversely, LysoPtdIns is a mitogen in several systems, including thyroid-derived cell lines; but to exert its function it needs to be released. However, there was no detectable release of LysoPtdIns during ATP and NE stimulation in these PCCl₃ cells. Increases in LysoPtdIns here thus support the formation of GroPIns due to receptor-activated PLA₂IV acting on membrane PtdIns rather than supporting a role for LysoPtdIns itself in cell growth control.

Direct addition of arachidonic acid stimulated cell proliferation but did not correlate with the role of adrenergic and purinergic receptors in this cell system. Indeed, while these receptors induce the same extent of cell growth, which correlates well with similar extents of GroPIns and LysoPtdIns production, the arachidonic acid increase is dissociated from this regulation; it is more pronounced after purinergic stimulation (100 µM ATP, 18-fold increase) than adrenergic stimulation (2-fold increase). Along with the relatively small effect on cell proliferation (2-fold increase) associated with the 30-fold increase in arachidonic acid release produced by the calcium ionophore A23187, this indicates that specific GroPIns production, rather than general arachidonic acid release, mediates receptor-activated PLA₂IV involvement in the control of cell proliferation.

We describe a novel pathway of cell growth regulation: receptor-induced activation of PLA₂IV and specific hydrolysis of membrane PtdIns to form GroPIns. Thus, PLA₂IV specificity in living cells is also at the level of substrate identification and the concomitant release of specific metabolites that control cell proliferation. These findings extent the regulatory pathways controlled by PIs metabolism.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5397fje

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