RASSF9 Regulated Epidermal Homeostasis Figure 11. RASSF9 expression profiles in WT mouse. (A) Quantitive RT-PCR analysis of RASSF9 mRNA expression in various tissues of WT mice at one and two weeks old. The results were normalized with regard to the mRNA expression of b-Actin (top panel), GAPDH (middle panel), or HPRT1 (bottom panel). Values represent the mean 6 SD (one-week-old mice, n = 4; two-week-old mice, n = 5). (B) Quantitive RT-PCR analysis of RASSF9 (top panel), E-cadherin (CDH1) (middle panel), and fibronectin (fn1) (bottom panel) mRNA expression in the epidermis and dermis of the WT skin at 4-dpp old. The results were normalized to individual reference gene of b-Actin, GAPDH, or HPRT1 (the reference gene is specified at the bottom). The results were normalized with regard to the mRNA expression of b-Actin, GAPDH, or HPRT1. Values represent the mean 6 SD (n = 4). *P,0.05, **P,0.001. (C) Frozen sections of 4-dpp skins were double-stained for RASSF9 (Red) and K1 (Green), with merged signals in yellow. ‘‘NC’’, negative control, with no incubation in anti-RASSF9 anti-serum; immunostaining procedures for NC were otherwise identical to ‘‘anti-RASSF9’’ panels. Blue, DAPI staining of cell nuclei. Scale bar = 100 mm. Area in white frame was enlarged to show the RASSF9 location in the WT skin (upper right panel). The relative intensity of RASSF9 in various epidermal layers of WT skin at 4-dpp were normalized to basal-layer signals (lower right panel). Data represent analyses by ImageJ of five individual images from each mouse (n = 3) and are presented as Mean 6SD fold intensity relative to basal layer. *, P,0.05. B, basal layer; S, suprabasal layer; G, granular layer. (D) Higher-magnification images of K1 (green) and RASSF9 (red) co-staining in frozen sections of dorsal skins of WT (+/+) and RASSF92/2 (2/2) mice. No RASSF9 signal was detected in RASSF92/2 epidermis. The dashed white lines denote the epidermis-dermis border. Similar results were obtained from three independent pairs of mice. Scale bar = 20 mm. Epi, epidermis; Der, dermis. doi:10.1371/journal.pone.0017867.g011 Discussion The N-terminal (NT) RASSF protein family is an evolutionarily conserved group that has orthologs in the lower vertebrate, Xenopus, and the invertebrate, Drosophila [1,4]; these genes are believed to potentially function in development. We first found that RASSF9 was predominately expressed in the stratified epithelium, where its function appeared to be linked to epidermal homeostasis. Indeed, absence of RASSF9 expression resulted in atypical histological architecture of the skin of RASSF92/2 mice, characterized by changes in thickness of histological architecture and abnormal development of hair follicles. In addition, aberrant distributions of various keratin markers for different stages of keratinocyte differentiation was observed in RASSF92/2 skin, indicating an inability to maintain a regulated program of epidermis development. The stratum corneum can provide a mechanical barrier to protect the body from the environmental damage and dehydra- tion; maintenance of epidermal development and homeostasis is thus critical for its proper function. The various anomalies observed in the skin of neonatal RASSF92/2 mice may therefore culminate in defective skin barrier function. In fact, assessment of skin permeability using the toluidine blue penetration assays showed that approximately 17% of heterozygous pups and 40% of homozygous pups exhibited moderately increased dye permeabil- ity of skin with a punctuated distribution of blue spots in RASSF92/2 mice versus WT control suggesting slightly compro- mised skin barrier for the RASSF9-deficient mice (Figure S6). In vitro study of the role of RASSF9 in epidermal keratinocyte proliferation and differentiation found that RASSF92/2 kerati- nocytes exhibited diminished or delayed response to differentiation induction by high calcium concentration, with attendant prefer- ence for hyperproliferation. Furthermore, adenovirus-mediated re- expression of RASSF9 could overcome those defects of the observed block in the terminal differentiation or hyperproliferation of RASSF92/2 cells. Based on these findings, we conclude that RASSF9 is critical for the regulation of skin epithelial proliferation and differentiation. To our knowledge, this is the first direct observation of the novel role of RASSF9 in maintaining homeostasis of cellular proliferation and differentiation. Although the RASSF9-knockout mice strain was generated by a transgenic insertion of the open-reading frame of an exogenous recombinant gene—EBV LMP-1—we believe that gene silencing by an intron disruption is not implausible. Various intronic regulatory elements have been found to be critical for regulation of gene expression [22,23,24,25,26]. In addition, the 7.2-kb intron sequence replaced by the transgenic insertion contains a 150-bps long sequence of purine-rich guanine-adenosine (GA) repeats possibly required for proper splicing of the primary transcript [27]. Indeed, we have consistently detected no presence of RASSF9 mRNA or protein expression in RASSF92/2 tissues. At the same time, neither mRNA transcripts of the LMP-1 transgene or its protein product was present at detectable level for the length of time frame in which the experiments were performed. Moreover, overexpression of LMP-1 alone by in vitro transduction of primary keratinocytes at a level drastically greater than the observed level in tissue samples resulted in no altered filaggrin expression levels (Figure S7). Even as it is likely that abrogated RASSF9 expression directly contributed to the observed phenotypes, the link between RASSF9 and the observed phenotypes may well be conclusively established by targeted knockout of the RASSF9 allele. The RASSF92/2 mouse line showed rapid death at two weeks old, and the histological analysis revealed a severe defect of pulmonary alveoli occurrence, marked by deficient septation of saccules that results in reduced number of alveolar sacs whose size are however grossly enlarged, as well as significant thickening of alveolar epithelium (Figure S8). Thus, RASSF9 was crucial for the maturation of organogenesis. The defective lung development of the RASSF92/2 mice is likely the major causality of their failure to thrive and premature death. Unexpectedly, we also observed a slight decrease in the survival rate of WT mice progeny of a heterozygous mother (+/2) before weaning that may be attributed to cachexia of the heterozygous mother versus healthy WT mother mice (Figure 1C). Consistent with this phenomenon, we observed normal pups born from the WT mother with a normal survival rate (data not shown). The terminal differentiation marker, filaggrin, which is normally expressed in the epidermal granular layer of WT skin, was detected in multiple layers in RASSF92/2 mice skin (Figure 10). However, in vitro-cultured RASSF92/2 keratinocytes were found to have significant defects or delays in terminal differentiation. This discrepancy could be explained by RASSF92/2 cells having an unusual proliferative capacity in vivo, which might cause multiple granular layers in epidermis by cell accumulation with a characteristic of delayed differentiation. RASSF9 is expressed throughout the entire epidermis of WT skin, which consists of the basal layer of growing keratinocytes and the suprabasal layer of differentiating keratinocytes (Figure 11C and D; Figure S4). This further supports our contention that RASSF9 plays a critical role in epidermal homeostasis. We showed that the re-expression of RASSF9 in RASSF92/2 keratinocytes could induce p21Cip1 expression and reduce DNA synthesis. Indeed, quantitative analysis of p21Cip1 (CDKN1A) mRNA expression in mouse skin tissue by QRT-PCR revealed a general decrease of p21Cip1 mRNA in the skin of RASSF92/2 mice (Figure S9). Additionally, we also attempted to determine the protein level of p21Cip1 in skin tissue by Western blot, but p21Cip1 protein was not detectable in skin tissue by this method because of less p21Cip1 protein and high nonspecific mask in there. Previous studies have shown that p21Cip1 inhibits DNA PLoS ONE | www.plosone.org 15 March 2011 | Volume 6 | Issue 3 | e17867