A Novel Role of RASSF9 in Maintaining Epidermal Homeostasis Chiou-Mei Lee1,2*, Polung Yang3, Lih-Chyang Chen2, Chia-Chun Chen3, Shinn-Chih Wu 4 , Hsiao-Yun Cheng 3 , Yu-Sun Chang2,3 1 Department of Medical Research and Development, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan, 2 Graduate Institutes of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, 3 Chang Gung Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, 4 Department of Animal Science and Technology, National Taiwan University, Taipei, Taiwan Abstract The physiological role of RASSF9, a member of the Ras-association domain family (RASSF), is currently unclear. Here, we report a mouse line in which an Epstein-Barr virus Latent Membrane Protein 1 (LMP1) transgene insertion has created a 7.2- kb chromosomal deletion, which abolished RASSF9 gene expression. The RASSF9-null mice exhibited interesting phenotypes that resembled human ageing, including growth retardation, short lifespan, less subcutaneous adipose layer and alopecia. In the wild-type mice, RASSF9 is predominantly expressed in the epidermal keratinocytes of skin, as determined by quantitative reverse-transcription PCR, immunofluorescence and in situ hybridization. In contrast, RASSF92/2 mice presented a dramatic change in epithelial organization of skin with increased proliferation and aberrant differentiation as detected by bromodeoxyuridine incorporation assays and immunofluorescence analyses. Furthermore, characteristic functions of RASSF92/2 versus wild type (WT) mouse primary keratinocytes showed significant proliferation linked to a reduction of p21Cip1 expression under growth or early differentiation conditions. Additionally, in RASSF92/2 keratinocytes there was a drastic down-modulation of terminal differentiation markers, which could be rescued by infection with a recombinant adenovirus, Adv/HA-RASSF9. Our results indicate a novel and significant role of RASSF9 in epidermal homeostasis. Citation: Lee C-M, Yang P, Chen L-C, Chen C-C, Wu S-C, et al. (2011) A Novel Role of RASSF9 in Maintaining Epidermal Homeostasis. PLoS ONE 6(3): e17867. doi:10.1371/journal.pone.0017867 Editor: Frank Beier, University of Western Ontario, Canada Received October 6, 2010; Accepted February 16, 2011; Published March 21, 2011 Copyright: 2011 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the grant from the Ministry of Education, Taiwan (to Chang Gung University), and grants from Chang Gung University and the Chang Gung Memorial Hospital (CMRPD 160053 and BMRP021 to YSC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lcm5132@mail.cgu.edu.tw Introduction The RASSF proteins comprise an evolutionarily conserved protein family of ten members (RASSF1 to RASSF10) in vertebrates [1]. RASSF1 through RASSF6 all harbor C- terminal Ras-association (RA) domains and are grouped together as the C-terminal RASSF family, while RASSF7 through RASSF10 all contain N-terminal RA domains and are grouped as the N-terminal RASSF family. The members of the N-terminal RASSF family lack the characteristic Sav–RASSF– Hpo (SARAH) domains found in members of the C-terminal RASSF family [2]. The C-terminal RASSFs, as well as RASSF8 and RASSF10 of the N-terminal RASSFs, have been reported to show reduced expression in various cancers, and are therefore considered to be tumor suppressors [2,3,4,5]. RASSF9 was originally named P-CIP1 (PAM C-terminal interactor 1) following its identification as a peptidylglycine-amidating monooxygenase (PAM)-trafficking protein, which has been linked to the recycling of endosomes [6] and interaction with N-, K- and R-Ras proteins in GST pull-down assays [7]. Although the gene encoding RASSF9 is known to be expressed in multiple organs, including testis, kidney, skeletal muscle, liver, lung, brain, and heart [6], its biological and physiological roles are not yet fully understood. Recently, we obtained an Epstein-Barr virus Latent Membrane Protein 1 (LMP1) transgene-insertion-derived mutant mouse line, which was a RASSF9 deficient mutant with phenotype typical of the alopecia syndrome. In normal skin, the proliferative cells are confined to a single basal layer, and the non-proliferative differentiating cells are located in the suprabasal layers. At the final stage of differentiation, the stratum corneum is formed at the outer layer of the epidermis, where it serves as a barrier that prevents epidermal water loss [8]. The stratum corneum is composed of a number of proteins, including loricrin, involucrin and filaggrin, all of which are associated with keratin interme- diate filaments [9]. Calcium-induced differentiation of primary mouse keratinocytes in culture provides a well-established model for the complex program of differentiation that occurs in vivo in the transition from the basal to upper epidermal layers [10]. Since a particular group of epithelial cells among epidermal epithelium is responsible for generating new hair follicle epithelium during each hair cycle [11,12], the intriguing phenotype of the mutant mice therefore prompted us to investigate the possibility that RASSF9 plays some important roles in regulating epidermal homeostasis. Here, we show that RASSF9 is predominantly expressed in epidermal keratinocytes of skin, and loss of RASSF9 expression results in hyperplasia and aberrant differentiation of epidermis. PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17867