RASSF9 Regulated Epidermal Homeostasis Figure 13. The effect of RASSF9 on p21Cip1 expression in keratinocytes. (A) RASSF92/2 and WT mouse keratinocytes were cultured in growth (0.06 mM calcium) and differentiation-inducing (2 mM calcium) medium for 2 days (left panel) and 4 days (right panel) prior to lysis and immunoblotting with the indicated antibody. K14-normalized intensity of p21Cip1 protein signal was determined by ImageJ software. The results were further normalized to baseline control (lane 1 of same blotting) and shown on the top of blot images. Mean 6 SD (n = 3). *, P,0.05; +/+, wild type; 2/2, RASSF92/2. (B) Expression by transduction of HA-RASSF9 or GFP as negative control in RASSF92/2 cells under the growth condition (0.06 mM calcium) was examined for indicated time points. Immunoblotting and data analysis were performed as described in (A). Fold intensities relative to respective GFP controls are shown on the top of blot images. Mean 6 SD (n = 2). *, P,0.05. (C) Re-expression of RASSF9 in RASSF92/2 cells under differentiation-inducing condition. Recombinant adenovirus infection, cell-lysate harvesting, immunoblotting and data analysis were performed as described in (B), except cells were subsequently incubated in 2 mM calcium medium for 2, 3 and 4 days on post transduction. Mean 6 SD (n = 3). doi:10.1371/journal.pone.0017867.g013 cytes and tissues by immunoblot were inconclusive. Nevertheless, we were able to confirm differential RASSF9 expression in skin tissues by other means of biochemistry and immunohistochemistry as described above. These findings suggest that RASSF9 gene induction is required for triggering keratinocyte growth arrest that facilitates subsequent differentiation. Although RASSF9 expression resulted in diminished filaggrin expression indicative of altered or delayed terminal differentiation in mouse primary keratinocytes, the disruption of a single allele (RASSF9+/2) was not sufficient to yield this characteristic (Figure S11). Notably, however, heterozygous (RASSF9+/2) mice showed signs of haploinsufficiency, with milder versions of the syndromes seen among the RASSF92/2 homozy- gotes (Figure 1A–C). Although overexpression of RASSFF9 by adenoviral transduction in RASSF92/2 keratinocytes efficiently rescued filaggrin expression at low multiplicity of infection (MOI = 5), increasing dosages of RASSF9 beyond 5 MOIs were not able to further enhance the filaggrin expression in RASSF92/2 keratinocytes (Figure S12A). Additionally, overexpression of RASSF9 by adenoviral transduction in WT (RASSF9+/+) mice keratinocytes also did not result in induction of filaggrin expression under high-calcium condition (Figure S12B). This saturating nature of RASSF9-induced filaggrin expression in mouse primary keratinocytes implies a limit on dependence of filaggrin induction on RASSF9, the intrinsic expression level of which is both necessary and sufficient for normal pattern of filaggrin expression. These findings indicate a possibility that RASSF9 may play an important, decisive role in facilitating the initiation of keratinocyte differenti- ation, rather than reinforcing it. While we observed altered proliferations and attendant anomalies in keratinocyte differentiation of RASSF92/2 tissues and cells in vitro and in vivo, the question remains on the precise nature of the alteration. It remains possible that RASSF9 deficiency results in altered preference for proliferation without damaging the machinery required for initiation and maintenance of the keratinocyte differentiation program, and the observed changes in selected differentiation markers such as filaggrin and loricrin represent the effect of this altered preference. Regardless, it is apparent that RASSF9 directly or indirectly enforces entry of keratinocyte differentiation, and in its absence cells fail to respond PLoS ONE | www.plosone.org 17 March 2011 | Volume 6 | Issue 3 | e17867