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 P2Y 2 and P2Y 6 receptors endogenously and determined the pathways by which the P2Y 2 controls proliferation and Na + /K + ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y 6 also controls proliferation and Na + /K + ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y 6 activation controlled cell proliferation but not the activity of the Na + /K + ATPase. UDP activation of P2Y 6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C‐α, ‐β, ‐δ, ‐ε, and ‐ζ but not of PKC‐ι and ‐η; (c) the phosphorylation of the extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c‐Fos protein. The P2Y 6 ‐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 P2Y 6 . 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‐ζ inhibition, suggesting that it may be due to PKC‐α/β and ‐ζ 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. © 2004 Wiley‐Liss, Inc.