C/EBPa and C/EBPb Are Required for Sebocyte Differentiation and Stratified Squamous Differentiation in Adult Mouse Skin John S. House1, Songyun Zhu 1 , Rakesh Ranjan1, Keith Linder2, Robert C. Smart1* 1 Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina, United States of America, 2 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America Abstract C/EBPa and C/EBPb are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPa and C/EBPb in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/ EBPa or C/EBPb alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPa and C/EBPb in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPa and C/EBPb in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R), two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPa and C/ EBPb are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal. Citation: House JS, Zhu S, Ranjan R, Linder K, Smart RC (2010) C/EBPa and C/EBPb Are Required for Sebocyte Differentiation and Stratified Squamous Differentiation in Adult Mouse Skin. PLoS ONE 5(3): e9837. doi:10.1371/journal.pone.0009837 Editor: Simon Williams, Texas Tech University Health Sciences Center, United States of America Received December 15, 2009; Accepted March 2, 2010; Published March 23, 2010 Copyright: 2010 House 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 work was supported by a grant from the National Cancer Institute (CA046637), a grant from the National Institute of Environmental Health Sciences (ES012473) and a training grant from the National Institute of Environmental Health Sciences (ES007046). 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: rcsmart@ncsu.edu Introduction There are six members of the CCAAT/enhancer binding protein (C/EBP) family: C/EBPa, C/EBPb, C/EBPc, C/EBPd, C/EBPe and C/EBPf. C/EBPs are members of the basic leucine zipper (bZIP) class of transcription factors. C/EBPs play important roles in fundamental cellular processes including proliferation, apoptosis, differentiation, inflammation, senescence and energy metabolism [1,2]. In terms of differentiation, C/EBPa plays a role in the differentiation of myeloid cells [3,4], alveolar type II cells [5], hepatocytes [6,7] and adipocytes [8–10] while C/EBPb has a role in the differentiation process in mammary epithelial cells [11,12], adipocytes [13], ovarian granulosa cells [14], and macrophages [15,16]. The interfollicular epidermis (IFE) of skin is a stratified squamous epithelium composed primarily of keratinocytes that undergo a highly coordinated program of sequential changes in gene expression as they migrate from the proliferating basal keratinocyte layer through morphologically distinct spinous and then granular suprabasal keratinocyte layers, ending in the production of a nonviable stratum corneum. A discrete population of IFE stem cells replenishes the basal cell compartment in the epidermis[17]. Epidermal homeostasis involves the intricate regulation and balance between proliferation of basal cells and their commitment to terminally differentiate. Notch signaling [18– 20] and AP-2a/AP-2c [21] have essential roles in epidermal development and postnatal epidermal differentiation. C/EBPa and C/EBPb have been shown to be highly expressed in mouse and human epidermis [22–24] suggesting a possible role for these transcription factors in stratified squamous differentia- tion. However, the analysis of an epidermal specific germline C/ EBPa knockout mouse revealed that C/EBPa is completely dispensable for epidermal homeostasis involving squamous differentiation and keratinocyte proliferation [25] and the analysis of germline C/EBPb knockout mouse epidermis revealed changes, albeit modest, in keratinocyte differentiation involving decreased K1 and K10 expression and increased basal keratinocytes proliferation, indicating a role for C/EBPb in the early events of stratified differentiation [26]. Because these changes in C/EBPb deficient epidermis were modest it led to the notion that C/EBPb has supportive rather than a major role in epidermal keratinocyte differentiation. However, another possibility for the lack of a major PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9837