RASSF9 Regulated Epidermal Homeostasis Figure 14. Schematic model of RASSF9-mediated maintenance of epidermal homeostasis through p21Cip1 in keratinocyte growth and differentiation. (A) In WT keratinocytes, RASSF9 expression is induced in cells progressing through early differentiation, but decreases thereafter as cells approach terminal differentiation. RASSF9 induces p21Cip1 expression in keratinocytes during growth and early differentiation, which may mediate in differentiation-related growth arrest. (B) A scheme illustrating that down-regulation of p21Cip1 expression, due to the loss of RASSF9 signaling in post-mitotic cells adjacent to the proliferation compartment, leads to the escape of keratinocytes from cell-cycle withdrawal and their subsequent proliferation. doi:10.1371/journal.pone.0017867.g014 properly to signals such as high calcium concentration, which is recognized as an inducer of keratinocyte differentiation [30,31,32,33]. Indeed, in addition to p21Cip1, our preliminary microarray analysis of the gene expression profile of RASSF92/2 keratinocytes hint at possible alteration of pathways known to be involved in epidermis development and keratinocyte differentia- tions. Further study would be necessary to delineate the exact mechanism by which RASSF9 governs the pathways and factors, including p21Cip1, that are required in concert for proper initiation of keratinocyte differentiation. Taken together, our main findings with regard to RASSF9 in keratinocyte growth and differentiation can be summarized as follows: 1) RASSF9 mRNA induction in WT keratinocytes was seen at the early stage of differentiation. 2) Overexpression of RASSF9 in RASSF92/2 keratinocytes can overcome the defects of differentiation program. 3) Overexpression of RASSF9 in RASSF92/2 keratinocytes inhibited hyperproliferation of the cells, which that linked to p21Cip1 induction under early growth conditions. The expression of p21Cip1 is known to be increased in cell populations adjacent to proliferation compartments (i.e., cells that are poised to differentiate). Furthermore, the protein is believed to be an important, p53-independent regulator of differentiation-associated growth arrest [20,34,35,36]. RASSF9 has been reported to associate with Ras proteins [7]. Ras proteins mediate multiple cellular functions, including cell proliferation, differentiation, survival, and apoptosis [37]. Over- expression of Ras in the suprabasal epidermis or skin equivalent has been reported to disrupt normal stratification, induce cell invasion and proliferation, and trigger the development of skin tumors [38,39,40]. Additionally, Ras may be associated with the morphology, spreading, growth, and differentiation of keratino- cytes [41,42,43,44]. However, high calcium-induced keratinocyte differentiation has been associated with down-regulation of Ras activity during the early stage of differentiation [45]. As we have observed that RASSF9 is transiently induced when 2 mM calcium is used to induce differentiation among keratinocytes, it would be interesting in the future to examine whether RASSF9 binds to endogenous Ras protein targets in keratinocytes under high- calcium treatment, leading to the attenuation of Ras activity during early differentiation in these cells. Based on our present findings, we propose a dynamic model of RASSF9 equilibration in keratinocytes at different differentiation stages (Figure 14). In this model, the profile of the endogenous RASSF9 gene expression is similar to that of the p21Cip1 expression with a dual pattern in primary keratinocyte differentiation, which was up-regulated by raising calcium to trigger early differentiation, but down-modulated in keratinocytes under terminal differentia- tion. Furthermore, we suggest that RASSF9 induces p21Cip1 expression in keratinocytes, which are then transitioned from the proliferative compartment to the growth-arrested early differenti- ation layer, where p21Cip1 functions in early differentiation-related growth arrest (Figure 14A). Conversely, the lack of RASSF9 PLoS ONE | www.plosone.org 18 March 2011 | Volume 6 | Issue 3 | e17867