RASSF9 Regulated Epidermal Homeostasis Figure 10. Aberrant differentiation in the skin of RASSF92/2 mice—filaggrin (green). (A) Frozen sections of dorsal skins of RASSF92/2 and WT control mice were immunostained with green fluorescence for filaggrin and red fluorescence for K14 in two-week-old mice. Blue, DAPI staining of cell nuclei. Scale bar = 100 mm. (B) Images of higher magnification for filaggrin staining (green fluorescence) in skin sections. The dashed white lines denote the epidermis-dermis border. Similar results were obtained from three independent pairs of mice. Scale bar = 20 mm; GL, granular layer; KL, keratinocyte layer. Note the increased thickness and patchiness in layer of prominent filaggrin staining, as well as abnormal distribution of filaggrin staining in keratinocyte layer and hair follicle (*). doi:10.1371/journal.pone.0017867.g010 RASSF9 modulates p21Cip1 induction in keratinocytes To gain insight into the potential involvement of cell-cycle regulation in the RASSF9-mediated growth of keratinocytes, we compared the Affymetrix microarray analysis data of gene expression patterns between RASSF92/2 and WT keratinocytes cultured in the growth medium. P21Cip1 (CDKN1A) was identified among several transcripts shown to be down-regulated in RASSF92/2 cells. The protein is a potent inhibitor of cyclin- dependent kinases and is known to be a key checkpoint protein for the transition of keratinocyte growth/differentiation [20]. To test whether the expression levels of p21Cip1 varied in keratinocytes cultured under growth (0.06 mM calcium) or differentiation-inducing (2 mM calcium) conditions, we examined p21Cip1 expression by Western blot analysis. Although the p21Cip1 level was significantly lower in RASSF92/2 keratino- cytes compared to WT keratinocytes when cells were cultured in the growth medium (0.06 mM calcium) for 2 or 4 days (approximately 60% and 50% of WT, respectively; Figure 13A), this difference gradually decreased when cells were cultured in the differentiation-inducing medium (2 mM calcium) from day 2 to day 4 (from 75% to 0%; Figure 13A). After 4 days of culture in the high-calcium medium, the cells further progressed toward the terminal differentiation stage; this was accompanied by a dramatic reduction of p21Cip1 protein levels in both RASSF92/2 and WT keratinocytes (Figure 13A, right panel). High calcium induces terminal differentiation of keratinocytes that is coincident with a more dramatic reduction of p21Cip1 [21], which can be used as an indicator of progression of keratinocytes toward terminal differentiation in this study. Thus, these results suggest that RASSF9 affects p21Cip1 expression primarily in keratinocytes that are at the stages of growth or early differentiation. To further test whether RASSF9 can modulate p21Cip1 expression in keratinocytes as they progress from growth to differentiation, we maintained in low-calcium condition RASSF92/2 keratinocytes transduced to express recombinant RASSF9. RASSF9 expression significantly induced p21Cip1 expression by 2.3-fold on day 1, but the fold-change diminished thereafter over time (Figure 13B). However, p21Cip1 induction in RASSF9-re-expressing RASSF92/2 cells did not occur under differentiation-inducing condition (Figure 13C). These results further support the hypothesis that RASSF9 affects p21Cip1 expression primarily in growth-phase or early-differentiating keratinocytes. Taken together, these findings strongly suggest that the reduction of p21Cip1 in RASSF92/2 keratinocytes potentially form the basis for the cellular hyperproliferation observed in the epidermis of RASSF92/2 mice. PLoS ONE | www.plosone.org 13 March 2011 | Volume 6 | Issue 3 | e17867