Differential signalling of purinoceptors in HeLa cells through the extracellular signal-regulated kinase and protein kinase C pathways.
We have previously shown that HeLa cells express P2Y2 and P2Y6 receptors endogenously and determined the pathways by which the P2Y2 controls proliferation and Na+/K+ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y6 also controls proliferation and Na+/K+ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y6 activation controlled cell proliferation but not the activity of the Na+/K+ATPase. UDP activation of P2Y6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C-alpha, -beta, -delta, -epsilon, and -zeta but not of PKC-iota and -eta; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c-Fos protein. The P2Y6 induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, thereby indicating that the ERK pathway mediates the mitogenic signalling of P2Y6. PKC and phosphoinositide 3-kinase (PI3K) inhibitors were tested at two different time points of ERK1/2 phosphorylation (10 and 60 min). The results suggest that novel PKCs and PI3K initiate the response but both conventional and atypical PKCs are required for the maintenance of the UDP-induced phosphorylation of ERK1/2. The induction of c-Fos was greatly diminished by conventional or atypical PKC-zeta inhibition, suggesting that it may be due to PKC-alpha/beta and -zeta activity. These observations demonstrate that UDP acts as a proliferative agent in HeLa cells activating multiple signalling pathways involving conventional, novel, and atypical PKCs, PI3K, and ERK. Of these pathways, conventional and atypical PKCs appear responsible for the induction of c-Fos, while ERK is responsible for cell proliferation and depends upon both novel and atypical PKCs and PI3K activities.
Muscella
Antonella
A
Laboratorio di Fisiologia Cellulare, Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Università di Lecce, Ecotekne, Lecce, Italia.
Greco
Simona
S
Elia
Maria Giovanna
MG
Storelli
Carlo
C
Marsigliante
Santo
S
D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003600:Cytosol; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects / Q000378:metabolism; D011983:Receptors, Purinergic / Q000378:metabolism*; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase; D014530:Uridine Diphosphate / Q000378:metabolism
D002118:Calcium; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007527:Isoenzymes; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D016760:Proto-Oncogene Proteins c-fos; D011983:Receptors, Purinergic; D000254:Sodium-Potassium-Exchanging ATPase; D014530:Uridine Diphosphate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one