The role of p27kip1 in the in vitro differentiation of murine keratinocytes.

We have studied the regulation of cyclins and cyclin-dependent kinase activities during differentiation of primary mouse keratinocytes. Differentiation was induced by placing primary murine keratinocytes into suspension culture, under conditions which prevent cells from attaching to any surface. This treatment induces synthesis of keratin 1, one of the earliest known markers of keratinocyte differentiation, and also results in a profound change in the regulation of G1 and S-phase cyclins and their associated proteins as well as their activities. The placement of cells in suspension culture reduced cyclin A, D1, and E kinase activity within 6 h, accompanied by the cessation of DNA synthesis. K1 mRNA levels were observed to increase after this period, supporting the hypothesis that cell cycle withdrawal precedes the differentiation program. Our data further revealed that the p27kip1 protein level and associated cyclin-dependent kinase inhibitory activity increased when keratinocytes were induced to differentiate. Pretreatment of adherent keratinocytes with p27kip1 antisense oligonucleotides dramatically reduced the accumulation of p27kip1 protein upon subsequent suspension culturing and prevented the onset of differentiation independently of the loss of cyclin-dependent kinase activities. Although antisense oligonucleotide treatment inhibited differentiation, it did not prevent growth arrest. Therefore, the differentiation of primary mouse keratinocytes required a function of Kip other than the inhibition of cyclin-associated activities, and we suggest that this requirement may reflect a novel Rb kinase activity present in Kip immune complexes, which is dependent on the presence of cyclin D3. Thus, the placement of keratinocytes in suspension induces a program that includes loss of cyclin activity, which is linked to terminal growth arrest, and an induction of p27kip1, which is linked to the differentiation program.