Keratin 8 Is Required for the Maintenance of Architectural Structure in Thymus Epithelium

et al. (2013) Keratin 8 Is Required for the Maintenance of Architectural Structure in Thymus Epithelium. PLoS ONE 8(9): e75101. doi:10.1371/journal.pone.0075101 Keratin 8 Is Required for the Maintenance of Architectural Structure in Thymus Epithelium Chikako Odaka Anne Loranger Kazuya Takizawa Michel Ouellet Michel J. Tremblay Shigeo Murata Akihito Inoko Masaki Inagaki Normand Marceau Shree Ram Singh, National Cancer Institute, United States of America Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/ K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity. - Funding: This work was supported in part by grants from the National Sciences and Engineering Council of Canada (312270) and the Canadian Institutes of Health Research (MOP15529). 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. Keratins (Ks), the intermediate filament (IF) proteins of epithelial cells, constitute a multigene family of acidic (or type I, K9 to K23) and basic proteins (or type II, K1 to K8 and K71 to K80), expressed as distinct pairs in a cell differentiation-dependent manner [1,2]. For instance, the K5/K14 pair is present in the keratinocytes of the basal layer of stratified epithelia, whereas the terminally differentiated keratinocytes contain the K1/K10 pair. K8 and its partner K18 are the first IF proteins expressed in the embryo [36], where they become prominent in simple epithelium and periderm at the late stage, and then restricted to simple singlelayered epithelia, such as lung and liver, at the post-natal stage [7]. Much of our knowledge on the multiple functions of K8/K18 IFs has derived from studies performed on K8-null mice generated via a targeted mutation in the germ line [8,9]. Actually, a large part of the work has used hepatocytes as cell model, given that their IFs consist solely of the K8/K18 pair and that a loss of one keratin partner normally leads to the degradation of the other [10]. For instance, using cultured K8-knockout (K8-null) mouse hepatocytes and their wild-type (WT) counterparts, we have demonstrated that K8/K18 IFs contribute to the maintenance of the integrity of the hepatocyte surface membrane in response to mechanical stress [11], and modulate the hepatocyte response to various apoptotic stimuli [1214]. Moreover, other lines of work on different K8-null versus WT mouse simple epithelial cells, such as those from intestine and pancreas, have revealed a K8/K18 IF involvement in epithelial cell polarity and protein targeting to subcellular compartments [1516], in spite of the fact that such cell types contain the K8/K18 pair in combination with 12 other keratins. In this context, it appears that the K8/K18 pair may exert multiple functions within the same epithelial cell type. Thymic epithelial cells (TECs) constitute a major component of thymic stroma, which provides a specialized microenvironment for survival, proliferation and differentiation of thymocytes [17]. Notably, TECs comprise two main types, referred to as cortical TECs (cTECs) and medullary TECs (mTECs), according to their regional confinement within the organ. Previous studies using Ks as markers, have revealed a K8 expression largely restricted to cTECs, with a small subset within the medulla versus a K5 expression mainly confined to the medulla, with a subset of K5+K8+ cells at the corticomedullary boundary and a scattered distribution in the cortex. Such differential keratin expression patterns have previously led to the proposal that K5+K8+ TECs constitute a population of immediate precursors to K5K8+ cTECs [18], in line with recent studies indicating that both types of TECs arise through differentiation of a common progenitor Figure 1. Keratin 8-deficient mice are absent from keratin 8/18 in thymic cortex. Immunofluorescence staining of thymus sections of 7week-old wild-type FVB/N mice (WT) and K8-deficient FVB/N mice (K8 KO) was performed to detect K8 (A) or K18 (B) (green) and K5 (A) or K14 (B) (red). K8-deficient mice are lack of K8/K18 expression in the cortex. Note the altered morphology of K5+ or K14+ mTECs in K8-deficient mice. Data are representative of independent experiments (n = 8 in each group). C, cortex; M, medulla. Scale bars = 100 mm. (C) Thymi from wild-type FVB/N mice and K8-deficient FVB/N mice at 5 weeks and 9 weeks of age were weighed. Each point provides the thymus weight of individual animal. doi:10.1371/journal.pone.0075101.g001 [19,20]; however, the keratin contribution to such a TEC differentiation is unknown. In work reported here, we used K8-null and WT mice to address the involvement of K8/K18 IFs in the cellular organization and integrity of thymic epithelium, in terms of cTEC and mTEC structure and organization, and mTEC differentiation status. The results show that K8/K18 IFs are required for the prevention of apoptosis in cTECs and the maintenance of structural integrity in both TEC types. Materials and Methods Mice Details on the establishment of the K8-deficient FVB/N mouse line have been reported previously [9]. K8-null mice and their WT littermates, in an FVB/N background, were housed in the specific pathogen free animal facility at our research center. Age- and sexmatched mice were used throughout the work. The Animal Care and Use Committee of the Centre de Recherche du CHU de Quebec approved the animal experiments. Experiments were performed according to the rules of our institutional animal care and use committee. Immunofluorescence Histology Thymus tissues were isolated and immersed in OCT compound. Frozen thymic sections were prepared (...truncated)