Divergent responses of ras-transfected and non-ras-transfected human keratinocytes to extracellular calcium.
Raising extracellular calcium (Ca(o)) induces terminal differentiation in cultured epidermal keratinocytes. The introduction of the ras oncogene into keratinocytes results in resistance to Ca(o)-mediated differentiation. To understand the signaling mechanism involved, we examined the Ca(o)-induced formation of inositol triphosphate (IP3) and changes in intracellular Ca2+ (Ca(i)) concentration in non-ras-transfected and ras-transfected HaCaT lines of human keratinocytes. When switched from 0.05- to 1.5-mM Ca(o) medium, the non-ras HaCaT line showed a rapid twofold increase in IP3 formation, whereas the IP3 level in the ras-transfected I-7 line was slightly affected. G-protein-coupled activation of phospholipase was intact in both lines, as evidenced by the generation of similar amounts of IP3 in response to addition of bradykinin or guanosine 5'-[gamma-thio]-triphosphate. Addition of 1.0 mM Ca(o) evoked similar Ca(i) responses in both non-ras- and ras-transfected cells: a transient elevation, followed by a sustained lower plateau. However, the two lines differed in their later responses: after being maintained in 1.0 mM Ca2+ for 24 h, the Ca(i) level was significantly lower in ras-transfected cells than in non-ras-transfected HaCaT cells. The Ca(o)-induced increase in Ca(i) in both lines was inhibited by the Ca2+ entry blocker SK&F 96365 or depolarization in high K+ bathing solution, demonstrating its dependence of calcium influx. The results suggest fundamental differences in the early signal that are generated in response to an increase in Ca(o) in ras-transfected keratinocytes, with the absence of a Ca(o)-induced rise in IP3--a signaling pathway defect that may play a role in the differentiation block the cells exhibit. In addition, the inability of ras-transfected cells to sustain a prolonged Ca(i) plateau may also contribute to their inability to differentiate in response to the Ca(o) signal.
Shi
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Department of Dermatology, University of California Davis, School of Medicine, 95616, USA.
Isseroff
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D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D011905:Genes, ras / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D011869:Radioligand Assay; D015398:Signal Transduction; D014162:Transfection
D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D001920:Bradykinin; D002118:Calcium