Divergent responses ofras-transfected and non-ras-transfected human keratinocytes to extracellular calcium

Abstract

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.