Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells.

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by Gö6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When Gö6976 and PD098059, or Gö6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or Gö6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.

Muscella A ，Elia MG ，Greco S ，Storelli C ，Marsigliante S ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

Effects of extracellular nucleotides in the thyroid: P2Y2 receptor-mediated ERK1/2 activation and c-Fos induction in PC Cl3 cells.

Elia MG ，Muscella A ，Romano S ，Greco S ，Di Jeso B ，Verri T ，Storelli C ，Marsigliante S ... -  《CELLULAR SIGNALLING》

PKC-zeta is required for angiotensin II-induced activation of ERK and synthesis of C-FOS in MCF-7 cells.

We examined the signalling pathways responsible for the Ang II induction of growth in MCF-7 human breast cancer cells. Ang II in MCF-7 cells induced: (a) the translocation from the cytosol to membrane and nucleus of atypical protein kinase C-zeta (PKC-zeta) but not of PKC-alpha, -delta, - epsilon and -eta; (b) the expression of c-fos mRNA and protein; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). All these effects were due to the activation of the Ang II type I receptor (AT1) since they were blocked by the AT1 antagonist losartan. The Ang II-stimulated ERK1/2 phosphorylation was blocked by (a) high doses of staurosporine, inhibitor of PKC-zeta, and by a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate region (zeta-PS); (b) PD098059, a mitogen-activated protein kinase kinase inhibitor (MAPKK/MEK); and, moreover, (c) the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin, thus indicating that PI3K may act upstream of ERK1/2. The Ang II-evoked c-fos induction was blocked only by high doses of staurosporine and by zeta-PS whilst PD098059, LY294002 and wortmannin were ineffective, thus indicating that c-fos induction is not due to ERK1/2 activity. When the epidermal growth factor-receptor (EGFR) tyrosine kinase activity was inhibited by the use of its inhibitor AG1478, Ang II was still able to induce ERK1/2 phosphorylation and c-fos expression, therefore proving that the transactivation of EGFR was not required for these Ang II effects in MCF-7 cells. The previously reported proliferation of MCF-7 cells induced by Ang II was blocked by PD098059 and by wortmannin in a dose-dependent manner, thereby indicating that in MCF-7 cells the PI3K and ERK pathways mediate the mitogenic signalling of AT1. Our results suggest that in MCF-7 cells Ang II activates multiple signalling pathways involving PKC-zeta, PI3K and MAPK; of these pathways only PKC-zeta appears responsible for the induction of c-fos.

Muscella A ，Greco S ，Elia MG ，Storelli C ，Marsigliante S ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

Mitogenic signalling by B2 bradykinin receptor in epithelial breast cells.

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain, and oedema. A role in the modulation or induction of healthy breast tissue growth has been postulated for tissue kallikrein present in human milk. Moreover, tissue kallikrein was found in malignant human breast tissue and bradykinin (BK) stimulates the proliferation of immortalised breast cancer cells. Aim of the present article was to investigate whether BK also exerts mitogenic activity in normal breast epithelial cells and partially characterise the signalling machinery involved. Results show that BK increased up to 2-fold the 24 h proliferation of breast epithelial cells in primary culture, and that the BK B2 receptor (not B1) inhibitor alone fully blocked the BK response. Intracellular effects of B2 stimulation were the following: (a) the increase of free intracellular Ca(2+) concentration by a mechanism dependent upon the phospholipase C (PLC) activity; (b) the cytosol-to-membrane translocation of conventional (PKC)-alpha and -beta isozymes, novel PKC-delta, -epsilon, and -eta isozymes; (c) the phosphorylation of the extracellular-regulated kinase 1 and 2 (ERK1/2); and (d) the stimulation of the expression of c-Fos protein. EGF, a well known stimulator of cell proliferation, regulated the proliferative response in human epithelial breast cells to the same extent of BK. The effects of BK on proliferation, ERK1/2 phosphorylation, and c-Fos expression were abolished by GF109203X, which inhibits PKC-delta isozyme. Conversely, Gö6976, an inhibitor of PKC-alpha and -beta isozymes, and the 18-h treatment of cells with PMA, that led to the complete down-regulation of PKC-alpha, -beta, -epsilon, and -eta, but not of PKC-delta, did not have any effect, thereby indicating that the PKC-delta mediates the mitogenic signalling of BK. Phosphoinositide 3-kinase (PI3K), tyrosine kinase of the epidermal growth factor receptor (EGFR), and mitogen activated protein kinase kinases (MEK) inhibitors were also tested. The results suggest that EGFR, PI3K, and ERK are required for the proliferative effects of BK. In addition, the BK induced cytosol-to-membrane translocation of PKC-delta was blocked by PI3K inhibition, suggesting that PI3K is upstream to PKC-delta. In conclusion, BK has mitogenic actions in cultured human epithelial breast cells; the activation of PKC-delta through B2 receptor acts in concert with ERK and PI3K pathways to induce cell proliferation.

Greco S ，Muscella A ，Elia MG ，Romano S ，Storelli C ，Marsigliante S ... -  《JOURNAL OF CELLULAR PHYSIOLOGY》

Extracellular ATP activates the PLC/PKC/ERK signaling pathway through the P2Y2 purinergic receptor leading to the induction of early growth response 1 expression and the inhibition of viability in human endometrial stromal cells.

Chang SJ ，Tzeng CR ，Lee YH ，Tai CJ ... -  《CELLULAR SIGNALLING》