Keratinocyte differentiation is associated with changes in the expression and regulation of phospholipase C isoenzymes.
In murine keratinocytes, Ca(++)-induced terminal differentiation is accompanied by a rapid and sustained increase of inositol phosphates and diacylglycerol. Based on Western blotting analysis, basal keratinocytes cultured in 0.05 mM Ca++ medium express phospholipase C (PLC)-gamma 1 predominantly and no detectable PLC-beta 1. Differentiating keratinocytes cultured in 1.4 mM Ca++ express two- to threefold more PLC-gamma 1 protein and PLC-delta 1, but no detectable PLC-beta 1. Although the amount of PLC-gamma 1 and -delta 1 protein increased, PLC-gamma 1 and -delta 1 mRNA decreased in differentiating cells. Thus the sustained rise of PLC activity induced by Ca++ in differentiating keratinocytes may be associated with higher amounts of both PLC-gamma 1 and -delta 1 in maturing cells, determined by a posttranscriptional mechanism. Tyrosine phosphate content in PLC-gamma 1 was low in basal cells and did not change in cells exposed to 1.4 mM Ca++. However, genistein inhibited the increase in PLC activity induced by 1.4 mM Ca++. In contrast, transforming growth factor (TGF)alpha, which stimulates both PLC activity and growth in basal keratinocytes, increased tyrosine phosphorylation of PLC-gamma 1. These results suggest that tyrosine phosphorylation of PLC-gamma 1 by the epidermal growth factor (EGF) receptor is linked to stimulated proliferation, whereas stimulation of PLC activity by Ca++ is linked to keratinocyte differentiation and involves the action of a tyrosine kinase but not tyrosine phosphorylation of PLC-gamma 1. Based on studies using the intracellular free Ca++ chelator BAPTA, a rise in intracellular free Ca++ was not required for stimulation of PLC activity by raising extracellular Ca++. Phorbol esters inhibited PLC stimulation by 1.4 mM Ca++ medium and increased serine phosphorylation of PLC-gamma 1. Exogenous phosphatidylinositol-specific and phosphatidylcholine-specific bacterial PLC also inhibited endogenous inositol phosphate formation and increased endogenous diacylglycerol (DAG). Thus, direct serine phosphorylation of PLC-gamma 1 by protein kinase C is associated with the inhibition of Ca(++)-mediated PLC stimulation. These results show that keratinocytes have multiple mechanisms to regulate PLC activity in response to a specific signal.
Punnonen
K
K
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda 20892.
Denning
M
M
Lee
E
E
Li
L
L
Rhee
S G
SG
Yuspa
S H
SH
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D007527:Isoenzymes; D014443:Tyrosine; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium