Abstract
MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium resulted in activation of Raf isozymes and their downstream effector ERK within 10-15 min, but did not increase JNK or p38 activity. Calcium-induced ERK activation differed in kinetics from mitogenic ERK activation by epidermal growth factor and could be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation led to down-regulation of Ras activity at the same time that ERK activation was initiated. Expression of a dominant-negative mutant of Ras also did not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin was sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation.
Highlights
The epidermis is a regenerative organ subject to permanent turnover throughout the life of an organism
Elevation of Extracellular Calcium Leads to a Rapid and Transient Activation of the Raf/MEK/ERK Pathway—To evaluate whether calcium-induced differentiation of keratinocytes is associated with activation of MAPK pathways, the spontaneously immortalized human keratinocyte cell line HaCaT [29] was stimulated by raising extracellular calcium levels from 0.05 to 1.0 mM, and activation of ERK, JNK, and p38 was analyzed in vitro by immune complex kinase assays
Calcium-induced Up-regulation of the Cyclin-dependent Kinase Inhibitor p21/Cip1 Is Sensitive to MEK Inhibition—Calcium stimulation resulted in a rapid ERK activation, which raises the possibility that the Raf/MEK/ERK pathway plays a role in the early stages of keratinocyte differentiation
Summary
The epidermis is a regenerative organ subject to permanent turnover throughout the life of an organism. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway.
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