The calcium signal for Balb/MK keratinocyte terminal differentiation induces sustained alterations in phosphoinositide metabolism without detectable protein kinase C activation.
Balb/MK keratinocytes require epidermal growth factor for proliferation and terminally differentiate in response to elevated extracellular Ca2+ concentrations. The molecular pathways controlling cell differentiation in this system have yet to be established. We show that a dramatic and sustained activation of phosphoinositide metabolism is produced upon addition of Ca2+ to Balb/MK cultures. The pattern of inositol trisphosphate isomers released in response to Ca2+ challenge appeared to be atypical. Inositol 1,3,4-trisphosphate release was observed by 30s and was produced earlier than any alteration in inositol 1,4,5-trisphosphate levels. Concomitant with the liberation of inositol phosphates, an increased production of diacylglycerol was observed. Despite a 3-fold increase in diacylglycerol levels detected even at 12 h after Ca2+ addition, no evidence of functional activation or down-regulation of protein kinase C was found. This was established by measuring p80 phosphorylation, epidermal growth factor binding, and protein kinase C levels by immunoblotting. Analysis of the diacylglycerol generated following Ca2+ addition to Balb/MK cells revealed that a significant proportion of that lipid was an alkyl ether glyceride molecular species. Therefore, it is possible that this diacylglycerol molecular species may play a role in the Ca2+-induced differentiation program of Balb/MK cells through mechanisms other than stimulation of classical protein kinase C.
Moscat
J
J
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Fleming
T P
TP
Molloy
C J
CJ
Lopez-Barahona
M
M
Aaronson
S A
SA
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016861:Cyclooxygenase Inhibitors; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D000078404:Epidermal Cells; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007294:Inositol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism; D007633:Keratins / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D016861:Cyclooxygenase Inhibitors; D004075:Diglycerides; D007295:Inositol Phosphates; D016859:Lipoxygenase Inhibitors; D010716:Phosphatidylinositols; D011480:Protease Inhibitors; D007294:Inositol; D004815:Epidermal Growth Factor; D007633:Keratins; D015544:Inositol 1,4,5-Trisphosphate; D066246:ErbB Receptors; D011493:Protein Kinase C; D010738:Type C Phospholipases; D005990:Glycerol; D002118:Calcium